Is Bhutan destined for 100% organic? Assessing the economy-wide effects of a large-scale conversion policy

L PLoS One. 2018; 13(6): e0199025. Published online 2018 Jun 13. doi: 10.1371/journal.pone.0199025 PMCID: PMC5999226 PMID: 29897989 Arndt Feuerbacher, Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft,1,* Jonas Luckmann, Conceptualization, Formal analysis, Methodology, Writing – review & editing,1 Ole Boysen, Conceptualization, Formal analysis, Methodology, Writing – review & editing,2 Sabine Zikeli, Conceptualization, Validation, Writing – review & editing,3 and Harald Grethe, Conceptualization, Methodology, Supervision, Validation, Writing – review & editing1 Anil Shrestha, Editor 1 International Agricultural Trade and Development Group, Humboldt-Universität zu Berlin, Berlin, Germany 2 School of Agriculture and Food Science, University College Dublin, Dublin, Ireland 3 Institute of Crop Science, Coordination for Organic Farming and Consumer Protection, University of Hohenheim, Stuttgart, Germany California State University Fresno, UNITED STATES Competing Interests: The authors have declared that no competing interests exist. * E-mail: ed.nilreb-uh@rehcabreuef.tdnra Author information ▼ Article notes ► Copyright and License information ► Disclaimer Go to: Abstract Organic agriculture (OA) is considered a strategy to make agriculture more sustainable. Bhutan has embraced the ambitious goal of becoming the world’s first 100% organic nation. By analysing recent on-farm data in Bhutan, we found organic crop yields on average to be 24% lower than conventional yields. Based on these yield gaps, we assess the effects of the 100% organic conversion policy by employing an economy-wide computable general equilibrium (CGE) model with detailed representation of Bhutan’s agricultural sector incorporating agroecological zones, crop nutrients, and field operations. Despite a low dependency on agrochemicals from the onset of this initiative, we find a considerable reduction in Bhutan’s GDP, substantial welfare losses, particularly for non-agricultural households, and adverse impacts on food security. The yield gap is the main driver for a strong decline in domestic agricultural production, which is largely compensated by increased food imports, resulting in a weakening of the country’s cereal self-sufficiency. Current organic by default farming practices in Bhutan are still underdeveloped and do not apply the systems approach of organic farming as defined in the IFOAM organic farming standards. This is reflected in the strong decline of nitrogen (N) availability to crops in our simulation and bears potential for increased yields in OA. Improvement of soil-fertility practices, e.g., the adoption of N-fixing crops, improved animal husbandry systems with increased provision of animal manure and access to markets with price premium for organic products could help to lower the economic cost of the large-scale conversion. Go to: Introduction There is an uncontested need to reduce the environmental impacts of agriculture. Converting from a conventional to an organic agricultural system is viewed as one strategy to meet this need [1]. A key challenge is, however, not to compromise the supply of food and biomass. Current studies on large-scale conversions vigorously debate the existence and size of yield gaps between conventional and organic agriculture [2] and the question of whether 100% organic agriculture (OA) can feed a growing world population [3–6]. So far, little research has been conducted on the economy-wide impacts of large-scale conversions to OA on a national scale. This might be due to the lack of genuinely formulated policy objectives, which aim to convert the entire agricultural sector on a large scale. Bhutan has received remarkable international attention following its announcement to become the world’s first 100% organic nation by 2020 [7].Besides environmental conservation, which is one of the four pillars of Bhutan’s Gross National Happiness philosophy, there are also other motifs for this policy, such as promoting the brand “Bhutan” [8]. OA is a holistic agricultural system that strictly relies on natural inputs and promotes practices like the maintenance of soil fertility, conservation of biodiversity, and animal welfare according to the norms of the International Federation of Organic Agriculture Movements (IFOAM) [9]. The usage of most chemical fertilizers (except for mineral phosphate and potassium not enriched by chemical processes), pesticides and genetically modified crops is prohibited. In animal husbandry, the use of artificial growth hormones is banned, and antibiotics are only allowed in exceptional cases. In 2012, 37% of Bhutan’s farmers used agrochemicals on about 19% of arable land [10]. Given its low reliance on agrochemicals, there is the notion that the country is destined for 100% OA [11]. However, this notion does not properly account for the difference between “organic by default” (i.e., no use of chemical inputs and mineral fertilisers) and organic by IFOAM principles, which are based on a holistic system approach [12]. Given the limited adoption of OA’s key principles, we refer to organic by default when using the term OA in the context of Bhutan. Due to the absence of agrochemical inputs in OA, access to nutrients for plant growth is limited and the occurrence of pests and diseases is more difficult to manage, resulting in lower yields than in conventional agriculture (CA). This is well documented for developed countries [2], but there is very limited research on OA’s relative productivity in developing countries generally, and in Bhutan specifically. Our study contributes to this research gap by estimating organic-to-conventional yield ratios of 16 annual and perennial crops using on-farm data from a nationally representative survey covering about 6,200 Bhutanese farmers. The main contribution of this study is to assess the economy-wide impacts of Bhutan’s proclaimed 100% organic policy. Given agriculture’s critical role for the country’s economy, employing about 50% of the labour force in 2012 [13], significant effects of such an unprecedented national conversion policy on household welfare and food self-sufficiency are to be expected. Furthermore, the detailed representation of the agricultural sector in our model allows to investigate how the conversion affects agricultural output, land use, and the availability of crop nutrients. Go to: Policy background The general concern for the environment is deeply anchored in Bhutan’s policies, for instance, by the constitutional mandate to maintain 60% forestry cover at all times [14] or the pledge to stay a carbon neutral country [15]. Part of the 100% organic policy rationale is to promote Bhutan as an organic brand, which shall help to commercialize smallholder agriculture, alleviate poverty, and add value to the tourism sector [16]. In order to become 100% organic, Bhutan’s government intends to “phase out [the] use of harmful chemical fertilizers and pesticides” [15], which effectively resembles a ban on agrochemicals. The use of mineral fertilizers compliant with IFOAM regulations (rock-phosphate and mineral potassium not enriched by chemical processes), do not play a role in Bhutan. Further, there is no domestic agrochemical production and the import of agrochemicals is a government controlled monopoly accounting for about 0.2% of total imports (in value terms) [17, 18]. Hence, a ban on importing or producing agrochemicals is relatively easy to implement and would only affect cropping systems directly, as animal husbandry in Bhutan follows traditional practices with negligible use of non-compliant inputs, such as artificial growth hormones. Since the first announcement of the 100% OA objective in 2008 [11], the consumption of synthetic fertilizers used in Bhutan remained stable, while the use of pesticides even increased with an average annual growth rate of 11.8%, which is attributed to labour shortages [19, 20] (see S1 Fig). In 2012, 13,943 hectares were cultivated under CA, i.e., 18.6% of total cultivated land [10]. Of the remaining 61,194 hectares only about 545 hectares of crop land (less than 1% of total arable land) were certified organic [21] leaving the largest share of cultivated land to organic by default, i.e., no organic certification and no usage of agrochemicals, but also no use of improved organic practices. However, the Royal Government of Bhutan (RGoB) does not necessarily tie achieving 100% OA to governmental certification, except for exports, for which an internationally recognized certification is compulsory [19]. The implementation of the 100% organic policy in Bhutan is very different from policy measures addressing organic farming, e.g., within the Common Agricultural Policy of the European Union (EU). In the EU, conversion to certified OA is based on incentives like subsidies compensating for yield reductions and increase of labour costs while rewarding environmental and societal benefits of OA. Clear regulations were developed in close relation to the IFOAM norms [9] that define organic agriculture and the practices applied (e.g., EC No 834/2007 [22]). A conversion to OA in such a context is usually a conscious decision made by farmers, who are motivated by reasons of economic viability and their ethical values and often these actions are embedded in grassroots-movements [23, 24]. However, expert interviews with government representatives, researchers, and farmers conducted between 2014 and 2016 showed that Bhutan’s 100% organic policy is not the result of a bottom-up process involving farmers, but rather a top-down policy. An early report even noted: “farmers lack awareness on organic farming. They are even confused: why organic now when they have just learnt to practice conventional farming” [16]. The top-down nature of Bhutan’s 100% OA policy merits special emphasis. It is largely based on the phasing out of chemical inputs, but does not properly integrate measures that lead to the adoption of improved organic farming practices (e.g., composting, improved manure management, use of nitrogen (N)-fixing plants, management of biodiversity for bio control and others) as defined in the principles of IFOAM [9]. CA farmers in Bhutan would thus predominantly convert to organic by default and not by IFOAM principles. The relevant stakeholders do not seem to sufficiently discuss and account for this difference. Bhutan, unlike many South Asian countries, has achieved a relatively high degree of food security as reflected by most nutrition indicators and in 2012, only 2.8% of the population were reported to live under the food poverty line of 2,124 kcal [25]. Yet, stunted growth in children younger than five years old and anaemia, which affects women and children, remain serious challenges [26]. Besides affecting food security, the OA policy could conflict with the objective of increasing self-sufficiency in cereals–a key priority of Bhutan’s agricultural policy. Through investments in mechanization and irrigation schemes, domestic paddy production shall be increased by 26% to achieve a rice-self-sufficiency rate of 67% until 2018 [8]. Considering the geopolitical tensions surrounding Bhutan, increasing food self-sufficiency is a valid national priority and this study will assess how this policy objective is affected by a 100% organic conversion. Go to: Materials and methods We simulated the 100% organic policy in Bhutan as a phase-out of agrochemical-use, according to which conventional farmers adopt the current farming practices of their organic by default counterparts without introducing any technological advancement. This simulation only concerned the cropping sector as the livestock sector does not use any inputs that are non-compliant with OA. We followed a two-step approach. In step one, we utilized data from a nationally representative farm survey to test for differences in productivity of OA and CA within each of Bhutan’s three agroecological zones (AEZs). In step two, we employed a single country Computable General Equilibrium (CGE) model to simulate the economy-wide impacts of the 100% organic policy. CGE models are a common method used to assess the effects of policies and economic shocks, particularly in the field of agriculture and food policy, as they comprehensively depict the interdependencies in the entire economy. Both steps are linked by updating the model’s database, a 2012 Social Accounting Matrix (SAM) of Bhutan, using the results of the yield difference estimation. Data and estimation procedure of OA-CA yield differences The analysis of yield differences is based on the nationally representative Agricultural Sample Survey (ASS) 2012 containing crop output data of about 6,200 farmers [10]. This dataset is the only recent survey that includes questions on agrochemical use in crops on a farm level. The dataset was obtained from the Ministry of Agriculture and Forests (MoAF) in Bhutan and was de-identified prior to access. Crops were classified as conventionally produced if farmers used chemical fertilizers, pesticides, or both on the crop area. The dataset was combined with altitude data on the sub-district (gewog) level to generate variables identifying the following three main AEZs. AEZ1 is the humid, sub-tropical zone at altitudes below 1,200 meters above sea level (masl). AEZ2 is the dry-subtropical AEZ in altitudes between 1,200 and 1,800 masl. AEZ3 is the temperate zone in altitudes above 1,800 masl. Yield data, calculated by dividing production in kilograms by area in hectares, was cleaned by excluding the 1% and 99% percentile outliers within each crop and AEZ. Yield differences were calculated as an organic-to-conventional ratio per crop and AEZ level if at least 25 observations were available per cultivation system (OA or CA), otherwise the crop was excluded from the analysis. Observations of crops cultivated without agrochemical use (i.e., organically-grown crops) by farmers who utilized agrochemicals in other crops were also excluded in order to avoid any possible effect of nutrients from synthetic fertilizers via crop rotations and to avoid any effects of pesticide application or pesticide drift. Fieller confidence-intervals were calculated for the yield ratios and significance levels were tested conducting the non-parametric Wilcoxon rank-sum test on absolute yield levels. A non-parametric procedure was applied as the Shapiro-Wilk W test revealed that the majority of crop yields neither follows a normal nor a lognormal distribution. The results of the yield gap analysis were utilized to update the model database. In case the yield difference was found to be significant (p-value < 5%), the cultivation system specific mean yields were used to compute total quantity produced per crop based on the distribution of land in the database among cultivation systems. If yield differences were not significant, the mean of all observations per crop across both cultivation systems was used. Economy-wide model framework Model database The 2012 SAM for Bhutan determines the structure of economic institutions and agents and is documented by Feuerbacher et al. [27]. The following two modifications were implemented: Firstly, data from the ASS 2012 and the Bhutan Living Standard Survey 2012 [13] were used to disaggregate farm activities, factors, and households according to the three AEZs. Crop-producing activities were further disaggregated according to cultivation systems (i.e., OA or CA), which results in cultivation system and AEZ specific input-output structures. Secondly, eight field operation activities (e.g., manual weeding with or without pesticide use) were incorporated using available crop budget data. Each produces a corresponding field operation commodity that enters the crop producing activity as a production input. The field operation “Mechanical land preparation”, for instance, comprises ploughing and puddling of land using a powertiller requiring fuel, agricultural labour (family and hired farm labour), and agricultural capital (powertiller). The incorporation of field operations is a novel feature in CGE modelling. It allows simulations to explicitly model the trade-off between technological choices and directly links the labour requirement to each operation, rather than aggregating it. Table 1 provides an overview of the SAM accounts. Farm activities comprise crop, livestock, post-harvest and community forestry activities and require farm production factors. Farm labour is either family labour (i.e., from the same household) or hired. We do not explicitly depict labour exchange groups, which is a common practice in Bhutanese agriculture, because the amount of labour supplied is typically close or equal to the labour received in return. There are two livestock accounts. Since other livestock than cattle (predominantly poultry, pigs, sheep, and goats) comprise only 5.2% of total livestock units in Bhutan [27], they are grouped together in the other animals account. In addition to farm labour and livestock there are also four agricultural land types, powertillers and other machinery. Income from these farm production factors accrues to either the farmer or landless households. We did not disaggregate by farm-size, which is rather homogenous in Bhutan, with more than 95% of farmers cultivating less than four hectares of land and a median landholding of about one hectare [10]. Non-agricultural households were differentiated by factor ownership and residence in either rural or urban areas. Agricultural households, activities and production factors were disaggregated by AEZ and in case of crop production and land also by cultivation system. In accordance with Bhutanese law, irrigated-land is only utilized by rice cultivation. Some activities have by-products such as crop residues, fodder, manure and draught animal services, which are used as inputs by other farm activities. Just as field operations, they are assumed to be non-tradable within Bhutan and thus were segmented according to AEZs. A table listing all SAM accounts in detail is provided in the supporting information (S1 Table). Table 1 Disaggregation of 2012 Bhutan SAM. Category Item Activities Agriculture: Crops1 (7), Livestock2 (2), Field operations1 (9), Community forestry1, Commercial forestry, Post-harvest processing1 (5) Industry: Extractive industries, Food processing, Textile, Other manufacturing, Public utilities Services: Construction, Trade, Transportation, Hotel and restaurants, Government services, Other services Factors Labour: Skilled, Unskilled, Family farm2, Hired farm2 Land: Rainfed land1, Irrigated land1, Orchards1, Pasture land2 Capital: Private capital, Public capital, Informal capital, Powertiller2, Other machinery, Cattle2, Other animals2 Margins Trade, Transportation Institutions Agricultural households: Farmer, Landless Non-agricultural households: Urban skilled, Urban unskilled, Rural skilled, Rural unskilled, Capital dependent, Transfer dependent Private enterprises, Public enterprises, Government, Rest of the world (Row) Taxes Import tax, Excise tax, Sales tax, Income tax, Production tax Capital Investments, Stock changes Open in a separate window Note: Number in parenthesis describes the quantity of items, e.g. there are seven crop sectors. 1 Disaggregated by the three agroecological zones and cultivation system 2 Disaggregated by the three agroecological zones Behavioural relationships in the model A CGE model captures the entire circular flow within an economy, as illustrated by the following example. A farmer household located in the low-altitude zone (AEZ1) allocates land and labour to an AEZ-specific cropping activity from which it receives a factor rent in return which finances its expenditure for consumption, taxes, transfers, and savings. The cropping activity receives income from the sale of its output, which is again needed to finance the use of intermediate inputs and factors. Hence, each agent or institution expenditure represents income somewhere else within the circular economy. According to our model setup, each crop can be produced by two cultivation systems (OA vs. CA) across the three AEZs and a Constant Elasticity of Substitution (CES) demand function is utilized to aggregate total supply across all activities producing the same crop. We assumed no price premiums for OA goods. Organic and conventional output are instead perfect substitutes produced with different technology and cost structures. There has been hardly any research assessing the Bhutanese consumers’ willingness to pay a price premium for OA produce. For the case of organic rice, no significant price premium was found to be paid by Bhutanese consumers [28]. Due to the lack of a domestic market for officially-certified organic products, this finding can be generalized for all crops that are produced under OA. Agricultural exports could potentially benefit from price premiums. Yet, except for a few minor export items, such as lemongrass oil, the vast majority of exports are not sold as organic products, even though cardamom and citrus fruits (two of the three most important agricultural export items) are produced without agrochemical use. Moreover, the purchasing power of the current main export markets, India and Bangladesh, which absorb 90% of total agricultural export value [18], is rather low and hence we do not assume a price premium for export goods. While the domestic market for organic products is not established yet, there is a strong awareness of Bhutanese consumers to distinguish between domestic and imported produce. This becomes apparent when visiting the country’s largest market, the Centenary Farmers Market in the capital city Thimphu, where Indian imports are sold at the ground floor and only Bhutanese products are allowed to be sold on the first floor. Using the Armington assumption [29] that makes imports imperfect substitutes for domestic products, we were able to reflect this pattern of consumer preference in our model. The CGE model adjusted for this study is the single country, comparative-static STAGE2 model which is comprehensively described in McDonald and Thierfelder [30]. The institutions and agents in the model are production activities, commodity markets, households, incorporated enterprises, the government, and the capital market. All markets are assumed to be perfectly competitive where consumers maximize utility and producers’ profits and prices adjust to clear markets. Household consumption follows the Linear Expenditure System according to which households maximize their utility from consumption of which a fixed part represents vital subsistence consumption.A large share of what Bhutanese farmers consume is their own produce. This is not explicitly reflected in the model. Instead, farmers purchase their own output through the commodity market. Activities describe the domestic production of commodities, which combine fixed shares of intermediate inputs (i.e., inputs required for the production process) and production factors (i.e., labour, capital and land) and transform them to outputs. In contrast to intermediate inputs, factor inputs can be substituted with each other to differing degrees (implemented as multi-level CES functions). The substitution possibilities between factors is determined by a production structure, which was extended to incorporate field operations. This novel approach allows to explicitly model technological trade-offs within the agricultural sector, such as land preparation using either manual labour, draught power or powertillers. It also includes a nest governing the substitution of cultivated land and fertilizer input, for which we use a low substitution elasticity of 0.4 as proposed by [31] in the context of developing countries. A detailed documentation of the model’s production structure and parameters is provided in the supporting information (S1 Text). Model closures Since Bhutan is a small country, the model assumes fixed world market prices. The external exchange rate is fixed. This reflects Bhutan’s current currency regime of a one-to-one peg of the Bhutanese Ngultrum with the Indian Rupee. India is by far Bhutan’s most important trade partner, accounting for more than 78% of imports and 94% of Bhutan’s exports in 2012 [18]. The consumer price index (CPI) is set as the model’s numeraire. The model is investment-driven (investment is fixed as a share of final demand) with equi-proportionately varying saving rates for households and enterprises. Government consumption and savings are fixed in quantity terms, and the government account is balanced by relative changes in the income tax rate. Capital supply is constant and assumed to be immobile and activity specific as we consider a short-term adjustment horizon. Skilled and unskilled labour are perfectly mobile across activities. Agricultural labour, both family and hired farm labour, are segmented according to AEZs, and thus only mobile within the activities of the same AEZ. The three land-types (irrigated land, rainfed land, and orchard) are set immobile across AEZs and cultivation systems, establishing the entry point of the shock of converting to organic agriculture. Unlike the other factors, the model closure for land accounts for unemployment, as a significant share of total arable land was left fallow in the base period. The land supply regime is explained within the next section. Modelling approach To achieve Bhutan’s 100% organic policy, a likely real-world policy instrument would be a ban on the use of agrochemicals. In a CGE model, such a policy is typically mimicked using a prohibitive tax (e.g., import or sales tax) which increases prices such that economic agents are incentivized to entirely substitute the use of the taxed good [32]. This approach has the disadvantage that a very high distortion of input prices would be needed in order to achieve a scenario close to 100% conversion. Therefore, a novel approach is applied in this study, modelling the phase-out of CA as a conversion of conventional to organic land within each AEZ and land type. Eq 1 describes the converted quantity of conventional to organic land CVT0on, which is the base supply of conventional land FS0cn multiplied by the share of conversion shrcvs and the diagonal matrix maplandcn,on to map organic (on) and conventional (cn) land-types according to AEZ and land type. Due to technical reasons, shrcvs is limited to 99.99% to simulate Bhutan’s 100% organic policy. CVT0on = FS0cn*shrcvs*maplandcn,on (1) Organic activities may not absorb all converted land and the portion of unutilized converted land is captured by the variable CVIon. Eq 2 formally describes the binary mechanism that determines the factor price WFon for organic land types (on). WFonCPI=⎧⎩⎨⎪⎪WF0onCPI,ifFWon≥FW0onorCVIon>0(FS0on+(FW0on−FWon)shfson)1elafson,ifFLWonCA depending on fertiliser inputs, management practices, crop species, and location. We therefore assume that under Bhutanese conditions similar context dependent results are to be expected. The only study assessing organic and conventional yields in Bhutan analysed rice grain yield data from 120 farms evenly sampled across all three AEZs in 2012 and 2013 [28]. Using ANOVA models the authors could not find any statistically significant differences between organic and conventional yields. This finding clearly differs from our results, as we find organic paddy yields in AEZ2 and AEZ3 to be 13% and 23% lower. Applying the same procedure (ANOVA) on our 2012 dataset, we find the yield difference of paddy even in AEZ1 to be statistically significant at the 1% level. The dataset used to estimate the yield gaps is the only representative farm survey conducted in Bhutan with crop specific questions on agrochemical use. However, it lacks further relevant information on variables that might influence yields, such as farmer’s education, asset ownership, intensity of input use as well as the slope of fields and the type of soils, which could have been used for regression analyses. To assess the robustness of our results with respect to any potential upward or downward biases of the yield estimates, we conducted a sensitivity analysis of the yield parameters. Discussion of model results Only a few studies have previously assessed the impacts of a large-scale conversion. Most of these are rather motivated by stylized thought-experiments using model approaches without accounting for economy-wide effects [42, 43]. The only comparable study simulates a 100% conversion for the Danish agricultural sector employing a comparative-static, single-country CGE model [44]. Under similar conditions, i.e., assuming no improvement in organic yields after conversion and allowing imports of animal feed, the authors find agricultural production to decline by -20.4% compared to the simulated decline of -10.6% for Bhutan. While organic farming practices might be more advanced in developed countries, there is also a greater dependency on agrochemicals, which could explain the comparatively lower drop in agricultural production for the case of Bhutan. Moreover, unlike developed countries, Bhutan’s livestock sector is predominantly ruminant based [45] and relies only to a small degree on conventional inputs and concentrate feed [46] and thus is only indirectly impacted by a 100% conversion. Looking at the economy-wide implications of a 100% OA conversion, the contraction in crop output leads to negative economic growth (-1.1% decline in real GDP or about 18.8 million USD). More than half of the reduction in crop output is due to the strong reduction in domestic production of paddy and potato, both of which are crops with large initial shares of conventional production and high yield gaps. Additionally, consumer preferences and trade linkages matter, as the example of the moderate decline in maize production shows. Trade also appears to buffer the drop in domestic production as reflected by the strong decline of the food self-sufficiency rate. In this context, the decline in paddy output appears to be most sensitive, which highlights the need to devote more resources to increase efficiency in organic paddy production, if the current degree of self-sufficiency is to be maintained. It is important to emphasize, that a 100% organic conversion on a country-level with the opportunity to import more conventionally produced food creates leakage of environmental externalities in the countries, which produce the additional imports. The reduction in GDP and household welfare is considerable given that the agricultural sector comprises only about 10% of GDP in 2012. From a distributional point of view, the cost of the 100% conversion would largely be borne by non-agricultural households. Among agricultural households particularly farmers in the temperate zone (AEZ3) would suffer from declining welfare. The welfare effects depend on the degree to which households are willing to substitute domestic food with imported food, since the price level of the latter is not impacted. The chosen model setup aggregates imports and domestic goods according to a national substitution elasticity, without considering household specific preferences. Referring to the four pillars of food security [47] our results show adverse impacts for food availability, access, and stability. Our model does not capture changes within the fourth pillar, food utilization (i.e., the metabolism of food by individuals) [48]. Food availability corresponds to the supply of food, which declines at the aggregate level by 3.6% and in case of crops by even 7.6%. Also, food access drops markedly as shown by the decrease in food consumption by households. Hence, the 100% OA policy is likely to partially reverse Bhutan’s past achievements in terms of food security and to jeopardize households close to or under the food poverty line of 2,124 Kcal. Our results show an increased dependency on food imports from India, which can have ambiguous effects on food stability. Stronger dependency on food imports might make the country less vulnerable against harvest failures within Bhutan, yet also increases the exposure to possible changes in India’s trade and foreign policy. From an agronomic perspective the model results are in line with ex-ante expectations regarding the impacts on land use and nutrient availability. Overall, the land area under cultivation increases, but only due to increased cultivation of rainfed land, while cultivation of orchards and irrigated land declines. In case of irrigated land, this is not only due to declining competitiveness of paddy cultivation, but also due to the Bhutanese law prohibiting other crops to be grown on irrigated land, which is a policy to foster rice-self-sufficiency. If other crops were allowed to be cultivated on irrigated-land, then paddy cultivation would likely decline further as irrigated-land would increase in price. Yet, in reality, many farmers cultivate other crops besides paddy on irrigated-land. Therefore, we probably underestimate the effects of the 100% organic policy on rice self-sufficiency. A key challenge of OA is the supply and availability of crop nutrients, particularly N. As the organic fertilizers (e.g., compost, animal manure) permitted in OA provide a lower nutrient density and a slower nutrient release compared to synthetic fertilizers, nutrient availability in OA often does not match plant needs [49]. Animal manure, predominantly from cattle, mixed with crop residues (i.e., farmyard manure) is the most important organic fertilizer in Bhutanese agriculture. Forests and grasslands account for about 22% and 53% of cattle’s feed requirement, respectively, allowing for a nutrient transfer to cropland [46, 50]. Yet, as cattle husbandry is often not stable based, animals either graze freely or are tethered on site. Thus, there is the potential for increased targeted nutrient transfer to crop land through improved animal husbandry systems, which has to comply with OA standards for animal welfare. After the simulated conversion, the availability of P and K remain stable or even increase. Animal manure cannot replace the previous share of N from synthetic fertilizers (27%), resulting in a decline in N availability (-22.4%). Our analysis assumes the initial headcount of livestock to be fixed, which limits the potential of additional manure production. In organic farming systems, the only source of N inputs that can replace synthetic N fertilizers is biological N fixation by cultivation of legumes (pulses, perennial legume based leys, or green manure in crop rotations) [51]. In the context of Bhutan, experiments have shown that green manuring is an option to achieve recommended nutrient levels in the soil for the cultivation of rice (but not for wheat) [35]. This potential, however, appears to be underutilized in Bhutan. Green manuring is rarely practiced [11] and pulses represent only about 3% of total cultivated area. Moreover, leguminous fodder crops are not yet used in Bhutan since most cattle husbandry systems are based on extensive grazing on pastures and in forests. In addition to legumes, further organic fertilizers such as leaf litter from forests and recycled materials from crop and household waste also play a rather minor role. Given the limited adoption of improved soil fertility management practices at the current stage and due to the lack of data, our model does not explicitly incorporate crop rotations and only includes manure as an organic fertilizer. Since multiple cropping and crop rotations are inherent features in the cropping system of both organic and conventional farmers in Bhutan [52], there is substantial potential to improve the adoption of N-fixing crops in order to lower the yield gap [53]. However, modelling their potential without sufficient data and recognizable efforts or initiatives undertaken to improve their adoption remains speculative. OA is generally known to be more labour intensive than CA, but this factor receives little attention in the literature. The labour intensity of organic rice production in Bhutan, for instance, is 11% higher for organic farmers due to higher labour requirement for weeding and applying farmyard manure [28]. Using the novel approach to model field operations, we explicitly depict the technological trade-off between both systems and show that aggregate labour absorbed by crop activities increases by 2.3%. Unlike claims in literature ([54, 55], our results do not show that the higher labour intensity of OA results in positive welfare effects for agricultural households, because of higher income from agricultural labour. Following the 100% OA policy, wages only increase slightly in those regions with low conventional production. In AEZ3, where the OA conversion is most challenging, agricultural output drops significantly and cultivated land declines by -3.4%, resulting in a lower demand for labour by cropping activities. Furthermore, in AEZ2 the benefit of slightly higher agricultural wages is offset by the loss in purchasing power, due to higher food prices. Since the OA conversion leads to higher labour requirement within specific periods, e.g., the periods of weeding and manuring, the depiction of seasonal labour would be intriguing and could influence changes in wages and land allocation. Yet, research on how to depict seasonality of labour markets in economy-wide models only started recently [56]. A further model limitation is the lack of labour mobility between agricultural and non-agricultural sectors. Non-agricultural wages are reduced at higher rates than agricultural wages, which implies that allowing migration between agriculture and non-agriculture sectors would result in an even higher decline of agricultural wages. Our model framework does not incorporate the environmental benefits from OA, due to a lack of data and research, which would help to derive their potential magnitude. The mentioned leakage-effect would also require to also considerations with regard to the adverse effects on countries from which additional conventionally produced food is imported. Moreover, our model does not account for any possible spillover effects that might improve Bhutan’s brand as a high-value tourist destination. However, according to the model results, tourist arrivals would need to more than double, if the societal cost of converting to organic farming should be born from an increase in tourist royalties. Sensitivity analysis In order to check the robustness of our model and results for variations in the yield gap, we simulated two further scenarios by varying the yield gap of all crops by +20% and -20%. Like in the reference scenario, this variation only affected those yield ratios that were found to be statistically significant. Following the described variation in yield gaps, real GDP varied only moderately, between -1.3% and 0.9% (Fig 5). The 20% increase in the yield gap triggered higher relative reductions in household consumption, food self-sufficiency, and agricultural production compared to a 20% decrease in the yield gap. This is due to the limited adaptive capacity of the agricultural sector to absorb a large reduction in its productivity. Manure production, for instance, cannot be increased infinitely and increases at a diminishing rate vis-à-vis the change in yield gap. In contrast, land cultivation increases (decreases) with a higher (lower) yield gap. Indeed, running an additional scenario without a yield gap, we even observe a drop in total land cultivation of -1.0%. Since agricultural labour and land are demanded in fixed shares, agricultural wages increase as more land is brought under cultivation. An external file that holds a picture, illustration, etc. Object name is pone.0199025.g005.jpg Fig 5 Sensitivity analysis to check robustness of main indicators for variation in the yield gap. Policy implications There is an obvious and serious conflict between the 100% OA policy with increased food self-sufficiency. In this paper, we assess this conflict in case of the current governmental approach of defining OA as “free of agrochemicals” while other aspects of OA being a holistic system are neglected [57]. Our study does not impose any adoption of improved organic farming practices, which have been proven to potentially close the yield gap in comparable situations [40], since there are no interventions or trends recognizable in Bhutanese agriculture that would make such an assumption reasonable. In case of an optimised organic farming system, the conflict between food self-sufficiency and OA may be reduced and it is an obvious policy implication, that this would require an adequate political strategy for the improvement of productivity of OA in Bhutan. In order to increase yield levels of today’s organic by default farming system, government investments in research and extension are necessary. Yet, up to now research on organic farming is concentrated at one of the five national Renewable Natural Resources (RNR) research centres [16] and the research policy formulated in 2011 only refers to organic farming once in 24 pages by stating to “give primacy to organically clean agriculture and minimize the role of external inputs” [58]. Given the challenge of converting to 100% organic, the current research capacity is still too limited and would need to be expanded significantly [19]. Specifically, technology development (e.g., optimised fertilization strategies based on biological N fixation, development of plant-based pesticides, adaptation of crop rotations, technological solutions for labour intensive practices such as weeding in rice, breeding of adapted crop varieties, improvement of mixed farming systems with animal husbandry) for an intensification of the traditional agricultural systems is needed to improve yields in OA. Besides research efforts and short-term dissemination of best-practice examples (peer-to-peer extension), demonstration farms and intensive training of extension workers could reduce the societal conversion costs. Access to export markets for high-value niche products might be another way to capitalize on the advantages of OA. Such opportunities could also arise in India in the long-run, where the awareness for quality and other added values of food products is growing among the middle class. However, the most important factor in the context of food self-sufficiency is the emergence of a market for organic produce inside Bhutan, which requires increased efforts to create consumer awareness. Other political strategies of dealing with the conflict of food self-sufficiency and OA are less powerful. For example, excluding the paddy sector to achieve higher rice self-sufficiency would largely diminish the benefits of an organic conversion and entail the risk of agrochemicals leaking into other cropping activities. Alternative to banning agrochemicals, taxing agrochemical inputs would reduce their usage where they are least efficient, while still allowing them where they provide a high benefit-cost ratio. In Bhutan, levels of agrochemical use are low, which can imply high marginal-benefits while causing low marginal-damage to the environment. Finally, such alternatives that aim at less than 100% OA will lower the potential of branding Bhutan as a genuine “organic and green country”, which may be considered the most important benefit of OA in Bhutan. Our results show the importance of increased conventional food imports to buffer the negative productivity shock on Bhutan’s cropping sector. This effect can hardly be avoided as any trade policy has to comply with Bhutan’s free trade agreement with India. A non-discriminatory measure would be to tax all agricultural goods that were not produced under organic standards, hence resulting in an effective import tax on most food items. However, this would further increase agricultural prices. A similar option would be to introduce sales taxes on agricultural produce, which could be rebated as production subsidies. This would lower the relative price difference between imported and local produce, but simultaneously provoke the risk of consumer protests in urban areas. As 100% organic is likely to benefit the tourism sector, policymakers could alternatively increase the tourism royalty in order to compensate farmers for lower productivity. This would benefit both farmers and consumers, yet the tourist’s willingness to accept such an increase in royalty is uncertain. Go to: Conclusions We studied Bhutan’s policy to convert 100% to OA analysing the economy-wide effects of such an unprecedented large-scale conversion policy. From a method development perspective, we conclude that shifting land between organic and non-organic farming activities in a CGE with a detailed depiction of the agricultural production structure, including the specification of complementary and substitutable field operations, is an adequate method to analyse the economy-wide effects of a conversion from conventional to organic agriculture. Regarding the implications of OA in Bhutan, we found a substantial yield gap between OA and CA for most crops despite the country’s low reliance on agrochemical inputs. We were able to show that lower yields and higher labour requirements in OA to substitute the previous use of pesticides and fertilizers in CA resulted in a strong contraction of agricultural output, substantial losses in welfare and negative implications for food security. Based on our data, converting Bhutan’s CA to the current organic by default farming system seems to be insufficient at fulfilling the promise of a 100% OA as a sustainable and environmentally friendly farming system that maintains current levels of food production. To reach this aim, further adaptations of cropping systems (fertilisation management, crop protection, integration of livestock) are necessary to narrow the current yield gaps and to develop truly holistic organic farming systems. We acknowledge that our database is far from sufficient to come up with conclusions regarding the potential of an optimized OA system, as little or no data for improved organic farming practices from field trials or farm pair comparisons exist for Bhutan. As for our model analysis, further research is needed to adequately address challenges that go beyond the scope of this study, such as potential environmental benefits and positive spillover effects, e.g., a boost in tourist arrivals. Against the background of substantial efforts by the Bhutanese government to make agriculture more productive and commercial, our results showed that the 100% organic policy affects the cropping sector like a production tax without the opportunity of redistributing any tax income. Bhutan’s policymakers should recognize these trade-offs, prioritize accordingly, and develop strategies to mitigate the conflict of objectives between OA and high food self-sufficiency. Increased efforts in the area of agricultural research, extension outreach and market development are necessary in order to ensure that a 100% OA policy can be reconciled with an improvement of rural livelihoods. If these efforts result in the adoption of advanced organic farming practices and access to price premiums then Bhutan’s 100% conversion to OA may result in lower economic cost than assessed in this study and farmers may even benefit from conversion to improved OA. Go to: Supporting information S1 Table Accounts of adopted 2012 Social Accounting Matrix. (PDF) Click here for additional data file.(50K, pdf) S1 Fig Use of agrochemicals in Bhutan 2005–2014. (PDF) Click here for additional data file.(207K, pdf) S2 Fig Percentage changes in livestock output, supply and purchaser prices after simulating a 100% organic policy. (PDF) Click here for additional data file.(122K, pdf) S1 Text Model extensions and parameters. (PDF) Click here for additional data file.(235K, pdf) Go to: Acknowledgments We are indebted to the academic editor, Anil Shrestha, and four anonymous referees for their valuable and constructive comments that helped to improve the article. We also thank Chencho Dukpa, Tulsi Gurung, Mahesh Ghimiray, Kesang Tshomo, Tiffany Williams, Lars-Bo Jacobsen, Scott McDonald, and Harvey Bradford as well as the participants at the 19th Global Economic Analysis conference in Washington DC, the 2016 Tropentag in Vienna and the 2016 DAAD Summer School at the College of Natural Resources in Lobesa, Bhutan for their helpful comments and feedback. Go to: Funding Statement The first author was funded by a PhD scholarship of the agricultural faculty at University of Hohenheim, Germany. We acknowledge the financial support of the fiat panis foundation that was received in form of a field research grant. Go to: Data Availability The underlying data of this study, the Agricultural Sample Survey 2012 and the 2012 Social Accounting Matrix for Bhutan, are third party data which the researchers obtained from the Ministry of Agriculture and Forests (MoAF) in Bhutan. 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Application of Bacteriocins and Protective Cultures in Dairy Food Preservation

Front Microbiol. 2018; 9: 594. Published online 2018 Apr 9. doi: 10.3389/fmicb.2018.00594 PMCID: PMC5900009 PMID: 29686652 Célia C. G. Silva,* Sofia P. M. Silva, and Susana C. Ribeiro Instituto de Investigação e Tecnologias Agrárias e do Ambiente, Universidade dos Açores, Angra do Heroísmo, Portugal Edited by: Riadh Hammami, University of Ottawa, Canada Reviewed by: M. Y. Sreenivasa, University of Mysore, India; Giuseppe Spano, University of Foggia, Italy; Ehab Eissa Kheadr, Alexandria University, Egypt *Correspondence: Célia C. G. Silva tp.cau@avlis.gc.ailec; tp.cau@ailec This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Author information ▼ Article notes ► Copyright and License information ► Disclaimer Go to: Abstract In the last years, consumers are becoming increasingly aware of the human health risk posed by the use of chemical preservatives in foods. In contrast, the increasing demand by the dairy industry to extend shelf-life and prevent spoilage of dairy products has appeal for new preservatives and new methods of conservation. Bacteriocins are antimicrobial peptides, which can be considered as safe since they can be easily degraded by proteolytic enzymes of the mammalian gastrointestinal tract. Also, most bacteriocin producers belong to lactic acid bacteria (LAB), a group that occurs naturally in foods and have a long history of safe use in dairy industry. Since they pose no health risk concerns, bacteriocins, either purified or excreted by bacteriocin producing strains, are a great alternative to the use of chemical preservatives in dairy products. Bacteriocins can be applied to dairy foods on a purified/crude form or as a bacteriocin-producing LAB as a part of fermentation process or as adjuvant culture. A number of applications of bacteriocins and bacteriocin-producing LAB have been reported to successful control pathogens in milk, yogurt, and cheeses. One of the more recent trends consists in the incorporation of bacteriocins, directly as purified or semi-purified form or in incorporation of bacteriocin-producing LAB into bioactive films and coatings, applied directly onto the food surfaces and packaging. This review is focused on recent developments and applications of bacteriocins and bacteriocin-producing LAB for reducing the microbiological spoilage and improve safety of dairy products. Keywords: bacteriocins, lactic acid bacteria, dairy products, biopreservation, edible coatings Go to: Introduction Bacteriocins are generally defined as peptides or proteins ribosomal synthesized by bacteria that inhibit or kill other related or unrelated microorganisms (Leroy and De Vuyst, 2004; Cotter et al., 2005). Bacteriocins may have a narrow spectrum, by inhibiting bacteria taxonomically close, or a broad spectrum, by inhibiting a wide variety of bacteria (Cotter et al., 2005; Mills et al., 2011). For the past years, bacteriocins have attracted considerable interest for their use as safe food preservatives, as they are easily digested by the human gastrointestinal tract (Mills et al., 2011). The use of bacteriocins as natural food preservatives fulfills consumer demands for high quality and safe foods without the use of chemical preservatives. However, the application of bacteriocins as food additives can be limited for various reasons, such as effectiveness of pathogen elimination or its high price (Chen and Hoover, 2003). Nevertheless, research interest in bacteriocins has continued over the past years, as investigators continuing to search for new and more effective bacteriocins to address both biologic and economic concerns. The application of bacteriocins for biopreservation of foods usually includes the following approaches: inoculation of food with the bacteriocin-producer strain; addition of purified or semi-purified bacteriocin as food additive; and use of a product previously fermented with a bacteriocin-producing strain as an ingredient in food processing (Chen and Hoover, 2003). An increasingly number of bacteriocins have been isolated and identified from Gram-positive and Gram-negative microorganisms. As a result, databases have been created to compile the information that can be used for the automated screening of bacteriocin gene clusters (Blin et al., 2013; van Heel et al., 2013). Classification of Bacteriocins For the past years, several classifications of bacteriocins have been proposed taking into consideration the first classification proposed by Klaenhammer (1993). Recently, in order to classify novel bacteriocins, Alvarez-Sieiro et al. (2016) proposed an adjusted classification scheme based on the biosynthesis mechanism and biological activity in accordance with other proposals (Arnison et al., 2013). They propose three major classes: Class I – small post-translationally modified peptides; Class II – unmodified bacteriocins; and Class III – larger peptides (>10 kDa, thermo-labile), being each one subdivided into subclasses. Mode of Action Bacteriocins have distinct mechanisms of action and can be divided into those that promote a bactericidal effect, with or without cell lysis, or bacteriostatic, inhibiting cell growth (da Silva Sabo et al., 2014). Most of the bacteriocins produced from LAB, in particular those inhibiting Gram-positive bacteria, exert their antibacterial effect by targeting the cell envelope-associated mechanisms (Cotter et al., 2013). Several lantibiotics and some class II bacteriocins target Lipid II, an intermediate in the peptidoglycan biosynthesis machinery within the bacterial cell envelope and, by this way they inhibit peptidoglycan synthesis (Breukink and de Kruijff, 2006). Other bacteriocins use Lipid II as a docking molecule to facilitate pore formation resulting in variation of the cytoplasm membrane potential and ultimately, cell death (Machaidze and Seelig, 2003). Nisin, the most studied lantibiotic, is capable of both mechanisms (Cotter et al., 2005). Some bacteriocins damage or kill target cells by binding to the cell envelope-associated mannose phosphotransferase system (Man-PTS) and subsequent formation of pores in the cell membrane (Cotter et al., 2013). Other bacteriocins can kill their target cells by inhibition of gene expression (Parks et al., 2007; Vincent and Morero, 2009) and protein production (Metlitskaya et al., 2006). Go to: Bacteriocins Produced by Lab Although there are several microorganisms that produce bacteriocins, those produced by the lactic acid bacteria (LAB) are of particular interest to the dairy industry (Egan et al., 2016). LAB have long been used in a variety of food fermentations by converting lactose to lactic acid, as well as producing additional antimicrobial molecules such as other organic acids, diacetyl, acetoin, hydrogen peroxide, antifungal peptides, and bacteriocins (Egan et al., 2016). As a result of their extensive use in traditional fermented products, most of the LAB are Generally Regarded as Safe (GRAS), granted by the American Food and Drug Agency (FDA). The European Food Safety Authority (EFSA) also granted the Qualified Presumption of Safety (QPS) status to most of the LAB genera, such as Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, and some Streptococcus (EFSA, 2007). Nevertheless, species of the genus Enterococcus and some Streptococcus are pathogenic, thus, they do not have GRAS status and were not proposed for QPS status (EFSA, 2007). Lactic acid bacteria bacteriocins are often active across a range of pH values, resistant to high temperatures and active against a range of food pathogenic and spoilage bacteria (Ahmad et al., 2017). In addition, LAB bacteriocins are sensitive to digestive proteases such as pancreatin complex, trypsin and chymotrypsin, and thus do not impact negatively on the gut microbiota (Egan et al., 2016). Go to: Application of Purified/Semi-Purified Bacteriocins to Dairy Products Bacteriocins have been used in the biopreservation of various foods, either alone or in combination with other methods of preservation, known as hurdle technology (De Vuyst and Leroy, 2007; Perez et al., 2014). Although results obtained from culture media may show that bacteriocins inhibit target organisms, the application of bacteriocins into foods must be tested to confirm their effectiveness. Many studies showed the potential of applying bacteriocins or bacteriocin-producing strains into foods, such as meat, dairy products, fish, alcoholic beverages, salads, and fermented vegetables (O’Sullivan et al., 2003; Ramu et al., 2015). To date, only nisin (Nisaplin, Danisco) and pediocin PA1 (MicrogardTM, ALTA 2431, Quest) have been commercialized as food preservatives (Simha et al., 2012). Although other LAB bacteriocins offered promising perspectives to be used as biopreservatives, as for instance the enterocin AS-48 (Sánchez-Hidalgo et al., 2011) or lacticin 3147 (Suda et al., 2012), no other bacteriocin has been proposed for industrial application. The screening of bacteriocins to be applied to foods requires the fulfillment of some important criteria. Producing strains should be food grade (GRAS or QPS), exhibit a broad spectrum of inhibition, present high specific activity, have no associated health risks, present beneficial effects (e.g., improve safety, quality, and flavor of foods), display heat and pH stability, and optimal solubility and stability for a particular food (Cotter et al., 2005; Leroy and De Vuyst, 2010). Various authors have reported that inactivation of several foodborne pathogens by bacteriocins may differ greatly depending on the food matrix used (Muñoz et al., 2007). Therefore, the effectiveness of different bacteriocins to foodborne pathogens must be tested in all food systems. Recent applications of bacteriocins into dairy foods to control food-borne pathogens included the inoculation of food with LAB that produce bacteriocins (Table ​Table11) or the addition of purified or semi-purified bacteriocins directly to food (Table ​Table22). Applying bacteriocin-producing LAB strains as antibacterial starter cultures and protective cultures may confer an advantage over the use of semi-purified/purified bacteriocins. In most cases, bacteriocins are adsorbed into food matrices and are easily degraded, which results in a loss of antibacterial activity. Therefore, an alternative method is the incorporation of bacteriocins into food packaging films/coatings, which improve their activity and stability in complex food systems (Salgado et al., 2015). Many researchers reported the potential of using immobilized bacteriocins in the development of antimicrobial packaging films to control food-borne pathogenic bacteria, such as L. monocytogenes (Sánchez-González et al., 2013; Ibarguren et al., 2015; Narsaiah et al., 2015). In fermented foods, the contamination with L. monocytogenes is a major concern. Several listeriosis outbreaks have been linked with the consumption of dairy products, in particular soft cheeses, causing a problem to dairy industry and public health authorities (Melo et al., 2015). Despite most dairy products, in particular cheeses, being made from pasteurized milk, contamination with Listeria still occurs. Cheeses are ready to use products and are usually conserved at refrigeration temperatures that allow the survival and growth of psychrotrophic bacteria, such as L. monocytogenes. Therefore, contamination can occur in later stages of dairy product processing (Carpentier and Cerf, 2011; Melo et al., 2015). Consequently, Listeria active bacteriocins emerge as an ideal solution for preventing the growth of this pathogen after cooking or packaging (Cotter et al., 2005). Bacteriocins can also be used to control adventitious non-starter flora, such as non-starter lactic acid bacteria (NSLAB) in cheese and wine, and in this way, contribute to the quality of the final product (Oumer et al., 2001; O’Sullivan et al., 2003). In addition, bacteriocins can also be used to enhance food fermentation, accelerating cheese ripening, and even improve its flavor (Oumer et al., 2001). Table 1 Applications of bacteriocin-producing LAB in dairy products (2000–present). Bacteriocin-producing strain Application technique Product Features Reference Staphylococcus equorum WS 2733 Cheese was inoculated with bacteriocin-producing strains Soft cheese L. monocytogenes growth was completely inhibited at low contamination levels and was reduced by 1-2 log at higher contamination levels. Carnio et al., 2000 Lactococcus lactis IFPL359 (transconjugant) Starter culture Goat’s milk cheese Acceleration of cheese ripening. Martinez-Cuesta et al., 2001 Lactococcus lactis CNRZ481 Adjunct culture Cheddar cheese Greater enzyme release through lacticin 481-induced lysis and reduction in the growth of NSLAB by 5 log. O’Sullivan et al., 2003 Enterococcus faecalis A-48-32 Adjunct culture Skimmed milk and non-fat hard cheese Bacillus cereus was reduced in milk to non-detectable levels after 72 h. In cheese, B. cereus decreased progressively, reaching 5.6 log after 30 days. Muñoz et al., 2004 Lactococcus lactis subsp. lactis INIA 415 Adjunct culture Hispánico cheese Acceleration of cheese ripening. Avila et al., 2005 Enterococcus faecium M241 and M249 Inoculation with bacteriocin-producing strains Skimmed milk Co-culture with E. faecium showed a delay in the growth L. monocytogenes of 6 h and reduced this pathogen by 2 log. Cocolin et al., 2007 Lactococcus lactis CL1 and CL2 (transconjugants, Nis+, Ped+) Adjunct to commercial starter culture Cheese L. monocytogenes was reduced by 2.97 (CL1) and 1.64 log (CL2). Rodriguez et al., 2005 Enterococcus faecium F58 Inoculation with bacteriocin-producing strain Jben (traditional fresh cheese) L. monocytogenes was reduced to below detection limits after 7 days of storage at 22°C. Achemchem et al., 2006 Enterococcus faecalis A-48-32 Inoculation with bacteriocin-producing strain Skimmed milk and fresh cheese Reduced Staphylococcus aureus, but efficacy was lower in cheese as S. aureus remained 1 log lower than control throughout storage. Muñoz et al., 2007 Lactococcus lactis Starter culture Cottage cheese Reduction of L. monocytogenes growth after 2 days, but a re-growth on days 5 and 7. Dal Bello et al., 2012 Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch Inoculation with bacteriocin-producing strains Fresh Minas cheese E. mundtii CRL35 displayed a bacteriostatic effect on L. monocytogenes up to 12 days at 8°C. E. faecium ST88Ch was less effective to control L. monocytogenes. Pingitore et al., 2012 Lactococcus lactis L3A21M1 and Enterococcus faecalis Inoculation with bacteriocin-producing strains Fresh cheese A combination of strains of E. faecalis reduced L. monocytogenes by 4 to 5 log after 7 days at 4°C. Enterococcus spp. were more effective in reducing L. monocytogenes in cheese. Coelho et al., 2014 Enterococcus durans E204 Adjunct culture Jben L. monocytogenes throughout the storage period and was undetectable after 6–8 days. Khay et al., 2014 Lactococcus lactis ssp. lactis CCMM / IAV/ BK1 Starter culture Jben L. monocytogenes was reduced to below the detectable level during storage at 7°C from days 1 to 6. Benkerroum et al., 2002 Lactobacillus sakei subsp. sakei 2a Cheese was inoculated with bacteriocin-producing strain Cheese spread L. monocytogenes was reduced at 4°C and decreased to below detection limit at 15°C, after 22–28 days Martinez et al., 2015 Lactobacillus plantarum CCDM 1078 Inoculation with bacteriocin-producing strains Cheese and quark based spreads L. monocytogenes was reduced by 1 log in both spreads. Patrovský et al., 2016 Staphylococcus equorum SE3 Inoculation with bacteriocin-producing strains Cheese model Inhibition of L. monocytogenes growth by 7 log units after 24 h. Bockelmann et al., 2017 Enterococcus faecium Cheese was inoculated with bacteriocin-producing strains Fresh whey cheese L. monocytogenes was reduced by 4 log after 6 days of storage at 4°C and was undetectable by the 9th day. Aspri et al., 2017 Enterococcus faecium KE82 Adjunct culture to commercial starter culture Raw and sterile milk L. monocytogenes was reduced by 4 to 5 log after 72 h at 22°C. Vandera et al., 2017 Lactococcus lactis (nisin producer) Starter culture Fresh cheese L. monocytogenes was reduced by 2 log after 7 days of storage at 4°C Kondrotiene et al., 2018 Open in a separate window Table 2 Applications of purified/semi-purified bacteriocins to control food-borne pathogens in dairy products (2000–present). Bacteriocin, producer Product Features Reference Nisin Z, Lactococcus lactis W8 Skim and whole-fat milk Reduced L. monocytogenes (5 log CFU ml-1) to undetectable levels in both skim and fat milk within 16 h at 8°C. Mitra et al., 2011 Nisin Z and A and lacticin 481, Lactococcus lactis Cottage cheese Cell-free supernatant (CFS) from the bacteriocin producing strains show weak ability to reduce L. monocytogenes Dal Bello et al., 2012 Nisin (commercial preparation, Nisaplin®) Galotyri PDO cheese Extended the shelf life of fresh Galotyri cheese stored at 4°C by 7 days. Kykkidou et al., 2007 Nisin A (Nisaplin®) Milk pudding Nisin A was effective to control spore-forming bacteria and extend shelf life. Reduced heat treatments to improve the flavor and aroma without compromising food safety. Oshima et al., 2014 Nisin (Nisaplin®) Minas Frescal cheese Nisin (500 IU⋅mL-1) reduced S. aureus in curd and whey, but the effect was minor during cheese storage. Felicio et al., 2015 Nisin, Lactococcus lactis N5764 Cow milk Combined nisin and phenolic compounds have a bacteriostatic effect on Staph. aureus and L. monocytogenes growth Alves et al., 2016 Lacticin 3147, Lactococcus lactis subsp. lactis DPC3147 Yogurt and cottage cheese Reduced L. monocytogenes to undetectable levels in yogurt within 10 min. In cottage cheese reduced 85% of L. monocytogenes contamination after 120 min. Morgan et al., 2001 Lacticin 481, Lactococcus lactis L3A21M1 Model fresh cheese Application of purified lacticin 481 reduced L. monocytogenes by 3 log after 3 to 7 days at 4°C. Ribeiro et al., 2016 Lactococcin BZ, L. lactis spp. lactis BZ and enterocin KP, E. faecalis KP Skim, half and full fat UHT milks Lactococcin BZ reduced L. monocytogenes in all milks to undetectable levels at 4°C or 20°C. Enterocin KP antilisterial result was affected by fat content. Yildirim et al., 2016 Leucocin K7, Leuconostoc mesenteroides K7 UHT whole-fat milk Combined leucocin K7 at 80 AU/ml and glycine at 5 mg/ml completely inhibited the growth of L. monocytogenes over 7 days Shi et al., 2016 Reuterin, Lactobacillus reuteri INIA PRO 137 UHT skim milk Combined reuterin and nisin reduced L. monocytogenes and S. aureus to undetectable levels after 12 days at refrigeration temperatures (4° and 8°C). Arqués et al., 2011 Variacin, Kocuria varians NCC 1482 Dairy food models Variacin (1% of fermented ingredient) was inhibitory to the growth of Bacillus cereus at 8°C, but this inhibition weakened as storage temperature increased. O’Mahony et al., 2001 Bovicin HC5, Streptococcus bovis HC5, and nisin Minas Frescal cheese Combined bovicin at and nisin at 600 AU g-1 completely inhibited the growth of L. monocytogenes after 9 days of storage at 4°C. Pimentel-Filho et al., 2014 Gassericins A and T, Lactobacillus gasseri LA39 and LA158 Custard cream The combined use of bacteriocins and glycine inhibited the growth of B. cereus. Arakawa et al., 2009 Aureocin A70, Staphylococcus aureus A70 UHT skim milk A partially purified aureocin preparation (16 AU mL-1) inhibited L. monocytogenes by 5.5 log at 4°C after 7 days. Fagundes et al., 2016 Enterocin CCM 4231, Enterococcus faecium CCM 4231 Saint-Paulin cheese Reduced L. monocytogenes by 4 log, but difference decreased to 1 log after 6 weeks of ripening. Lauková et al., 2001 Enterocin AS-48, Enterococcus faecalis A-48-32 Skimmed milk Combined with sub-lethal heat treatment (65°C for 5 min) reduced Staphylococcus aureus below detection limits. Muñoz et al., 2007 Ent35-MccV (hybrid bacteriocin), Escherichia coli BL21 Skim milk A complete elimination of E. coli was observed after 10 h at 37°C and 4 days at 4°C. L. monocytogenes was initially reduced but regrowth after 48 h at 37°C. At refrigeration (4°C), L. monocytogenes was reduced by 3 log after 10 days. Acuña et al., 2015 Enterocins, Enterococcus faecalis L3B1K3 Model fresh cheese Semi purified enterocin (536 μg/g) reduced L. monocytogenes to undetectable levels in cheese within 6 h. Ribeiro et al., 2017 Open in a separate window Over 230 bacteriocins produced by LAB have been isolated and reported, but only half of them were identified at the protein or DNA levels (Alvarez-Sieiro et al., 2016). Moreover, a limited number of purified or semi-purified bacteriocins have been tested in food systems, especially in dairy foods. Nisin The bacteriocin nisin is classified as a class-Ia bacteriocin or lantibiotic and is the most characterized and commercially important bacteriocin (Ross et al., 2002). Nisin is licensed as a food preservative (E234) and is recognized to be safe by the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives (FAO and WHO, 2006; Favaro et al., 2015). To date, eight types of nisin variants were discovered and characterized: nisins A, Z, F, and Q produced by Lactococcus lactis and nisins U, U2, P, and H produced by some Streptococcus strains (O’Connor et al., 2015). The commercially available form of nisin for use as a food preservative is NisaplinTM, with the active ingredient nisin A (2.5%) and other ingredients such as NaCl and non-fat dry milk (Chen and Hoover, 2003). Nisin has antimicrobial activity against numerous Gram-positive bacteria, including LAB, pathogens such as Listeria and Staphylococcus, and the spore forming bacteria, Bacillus and Clostridium (Chen and Hoover, 2003). One of the earliest applications of nisin was to prevent late blowing in cheese caused by gas-producing Clostridium spp. (Galvez et al., 2008). Nisin has been widely applied in cheese and pasteurized cheese spreads to replace nitrate for preventing the outgrowth of clostridia spores (Abee et al., 1995; Chen and Hoover, 2003). Nisin has also been shown to be effective in the control of different pathogens such as L. monocytogenes and Staphylococcus aureus in dairy products (Sobrino-López and Martín-Belloso, 2008). In a study of Arqués et al. (2011), nisin was shown to reduce L. monocytogenes and S. aureus in milk stored at refrigeration temperatures. Several studies also tested the addition of nisin to diverse cheese types (cottage cheese, cheddar, and ricotta-type cheeses) and show an effective reduction of L. monocytogenes growth, although limited to 1–3 log cycles (Chen and Hoover, 2003). Several reports indicate that nisin A is not very active against L. monocytogenes, but its anti-listerial effect is enhanced by the reduction of pH and addition of NaCl (Chen and Hoover, 2003; Khan and Oh, 2016). In Minas Frescal cheese, S. aureus counts were reduced by approximately 1.5 log cycles after addition of nisin (Felicio et al., 2015). In processed cheese, Bacillus cereus and Bacillus subtilis were also inhibited by nisin (Sobrino-López and Martín-Belloso, 2008). The use of nisin as an antimicrobial treatment extended the shelf-life of a Greek soft acid–curd cheese (Galotyri) by >21 days (Kykkidou et al., 2007). Incorporation of nisin (at a level of 2.5 mg l-1) has also shown to increase shelf-life of Ricotta-type cheese by inhibiting the growth of L. monocytogenes for >8 weeks (Davies et al., 1997). Moreover, a high level of retention of nisin was observed over the 10-week storage period, with only 10–32% of nisin loss. Ferreira and Lund (1996) also studied the effect of nisin on survival of the most resistant strains of L. monocytogenes in cottage cheese and showed a reduction to approximately 3 log cycles in 3 days. This bacteriocin was also tested in other pasteurized dairy products, such as chilled desserts, flavored milk, clotted cream, and canned evaporated milks, and was shown to reduce post process contaminating bacteria such as L. monocytogenes (Galvez et al., 2008). The efficacy of a combination of nisin and bovicin HC5 against L. monocytogenes and S. aureus in fresh cheese was studied by Pimentel-Filho et al. (2014). They observed a reduction of L. monocytogenes to undetected levels after 9 days of storage at 4°C, although the combination of bacteriocins did not prevent the growth of S. aureus. Several studies showed that the antimicrobial activity of nisin is affected by several factors including pH, temperature, composition, structure, and natural microbiota of food (Zhou et al., 2014). Proteolysis in cheese-making process may also affect the activity of nisin and limit its antimicrobial efficacy. Nevertheless, some authors described a limited loss in nisin activity (10–32%) in Ricotta cheese after 10 weeks of storage (Cleveland et al., 2001). Moreover, nisin activity was not affected by proteases in a study on Emmental cheese (Favaro et al., 2015). The application of nisin in dairy products is also limited to pH values lower than 7, as nisin greatly loses the activity at higher pH (de Arauz et al., 2009). Other studies also mention several limitations on the use of nisin in dairy products, due to its interaction with fat and other components in the food matrix (Favaro et al., 2015). However, the role of fat in the activity of nisin is not entirely clear, as studies on heat-treated cream show the inhibition of B. cereus growth by low concentrations of nisin (Nissen et al., 2001). Additionally, nisin was found to prevent spoilage bacteria and extend shelf-life in high-fat milk-based pudding (Oshima et al., 2014). In contrast, homogenization of milk was show to reduce the anti-listerial effects of nisin, demonstrating that the treatment of foods may play an important role in the efficacy of bacteriocins such as nisin (Bhatti et al., 2004). The wide spectrum of inhibition associated with nisin includes LAB themselves (Abee et al., 1995). Therefore, in dairy foods which require LAB for fermentation processes, the application of nisin presents a great limitation. An alternative consists in employing other bacteriocins with a highly specific activity range (Abee et al., 1995). As a result, there has been a great interest in the search for new bacteriocins with a widespread range of antibacterial activity, stability in different food environments, tolerance to heat, and resistance to proteolytic enzymes. Pediocins Pediocins are a class IIa bacteriocins produced by Pediococcus spp. and are commercially available under the name Alta 2341TM or MicrogardTM (Garsa et al., 2014). This bacteriocin has been shown to be more effective than nisin against some food-borne pathogens such as L. monocytogenes and S. aureus (Cintas et al., 1998; Eijsink et al., 1998) and Gram-negative organisms such as Pseudomonas and Escherichia coli (Jamuna and Jeevaratnam, 2004). The potential application of pediocins to dairy products is further enhanced by its stability in aqueous solutions, its wide pH range, and high resistance to heating or freezing (Sobrino-López and Martín-Belloso, 2008). Despite this high potential, few studies have investigated the addition of pediocins to milk or dairy foods. Pediocin (PA-1) was found to reduce L. monocytogenes counts in cottage cheese, cream, and cheese sauce (Pucci et al., 1988). The anti-listerial effect was noticeable over a wide temperature and pH ranges and was particularly effective at low initial L. monocytogenes contamination (102 cfu ml-1). Recently, Verma et al. (2017) described the production of food-grade pediocin from supplemented cheese whey medium. This semi-purified pediocin containing fermented cheese whey was shown to be effective in reducing S. aureus counts and enhancing shelf-life of raw buffalo milk (Verma et al., 2017). Lacticins Lacticins are produced by certain strains of Lc. lactis and comprise lacticin 3147 and lacticin 481 (Piard et al., 1992; McAuliffe et al., 1998). Lacticin 3147 was isolated from an Irish kefir grain used for making buttermilk and is a two-component lantibiotic, requiring both structural proteins to give full biological activity (McAuliffe et al., 1998). This bacteriocin exhibit antimicrobial activity against a wide range of food pathogenic and food spoilage bacteria in addition to other LAB (Sobrino-López and Martín-Belloso, 2008; Martínez-Cuesta et al., 2010). A lacticin 3147 powder preparation was shown to be effective for the control of Listeria and Bacillus in infant milk formulation, natural yogurt, and cottage cheese (Morgan et al., 2001). However, the high concentrations of lacticin powder used, which represent 10% of product weight, were considered impracticable and non-economic by the authors (Morgan et al., 2001). Lacticin 481 is a single-peptide lantibiotic that exhibits a medium spectrum of inhibition, mainly active against other LAB, Clostridium tyrobutyricum (O’Sullivan et al., 2003) and L. monocytogenes (Ribeiro et al., 2016). The use of non-purified lacticin 481 was show to have a mild bacteriostatic activity in milk stored at refrigeration temperatures (Arqués et al., 2011). Yet, the application of semi-purified lacticin 481 to fresh cheeses stored at refrigeration temperatures reduced L. monocytogenes by 3 log cycles in 3–7 days (Ribeiro et al., 2016). Nevertheless, the application of lacticins in food systems is not likely to ensure complete elimination of pathogens such as L. monocytogenes. Enterocins Enterocins are produced by Enterococcus species and comprise a diverse group of bacteriocins, both in terms of their classification and inhibitory spectrum (Egan et al., 2016). Although most LAB have the GRAS status and can be used safely in food applications, bacteriocinogenic enterococci raise some safety concerns (EFSA, 2007). Enterococci are among the pathogens that have been associated with a number of infections in humans (Moreno et al., 2006). In addition, some enterococci may harbor virulence determinants and antibiotic resistance genes (EFSA, 2007). While food enterococci have fewer virulence determinants than clinical strains, they are known for their capacity to exchange genetic information (Eaton and Gasson, 2001). Considering the safe concerns of using bacteriocinogenic enterococci, the use of purified enterocins may be considered more suitable for food consumption. Enterocin AS-48 is produced by Enterococcus faecalis and is a class IIc cyclic bacteriocin that is active against a number of Bacillus and Clostridium sp. (Egan et al., 2016). This is one of most studied bacteriocins, showing high stability to pH and heat, which makes a great candidate for application in food. Muñoz et al. (2007) tested the efficacy of the enterocin AS-48 for controlling staphylococci in skimmed milk. They observed a bactericidal effect proportional to the bacteriocin concentration (10–50 μg ml-1), but complete elimination of staphylococci was not achieved for any of the concentrations tested. This apparently lower effectiveness of AS-48 in milk when compared to culture medium could be attributed to a higher retention of the bacteriocin molecules by milk components and slower diffusion (Muñoz et al., 2007). Yildirim et al. (2016) investigated the antimicrobial effects of enterocin KP toward L. monocytogenes in skim, half fat, and full fat milks. Enterocin KP had a high anti-listerial effect, but this bactericidal effect decreased as both the fat content of milk and inoculation amount of L. monocytogenes increased. Also, Lauková and Czikková (1999) reported an inhibitory effect of purified enterocin CCM 4231 (3200 AU ml-1) on the growth of S. aureus and L. monocytogenes in skimmed milk and yogurt. Still, the antagonistic effect of enterocin on S. aureus in yogurt was less influential after 24 h. Usually, enterocins are active against foodborne pathogens, such as Listeria spp. and Clostridium spp., but their application in food systems is not likely to prevent the re-growth of pathogens throughout the storage time (Zacharof and Lovitt, 2012; de Souza Barbosa et al., 2015). However, Arqués et al. (2011) observed the reduction of L. monocytogenes counts in milk below the detection limit after 4 and 24 h by the combined effect of two bacteriocins, reuterin and enterocin AS-48. Recently, Ribeiro et al. (2017) reported the efficacy of a semi-purified enterocin produced by an E. faecalis strain, in reducing the contamination of L. monocytogenes in fresh cheese in a dose-dependent manner. Moreover, the highest dose applied to cheeses (approximately 2000 AU g-1 of cheese) resulted in reduction of this pathogen below detection levels and this effect remained for all the storage time (72 h). Other Bacteriocins Lactococcin BZ is produced by some strains of Lc. lactis spp. (lactis BZ) and has a wide antibacterial activity against Gram-positive and Gram-negative bacteria (Şahingil et al., 2011). This bacteriocin also displayed strong anti-listerial activity in milk. The partially purified lactococcin BZ (400–2500 AU ml-1) reduced L. monocytogenes counts to an undetectable level in both skim and full-fat milk during storage at 4 and 20°C (Yildirim et al., 2016). In addition, this anti-listerial activity was stable until the end of the storage period (25 days) and was not adversely affected by milk fat content. Aureocin A70 is a class II bacteriocin produced by a S. aureus strain isolated from pasteurized commercial milk (Fagundes et al., 2016). This bacteriocin exerted a bactericidal effect on a wide range of Gram-positive bacteria, including L. monocytogenes, and was tested in UHT-treated skimmed milk by Fagundes et al. (2016). Aureocin A70 caused a time-dependent reduction in L. monocytogenes counts (5.51 log units) up to 7 days of incubation, but was insufficient to achieve the total elimination of the pathogen, resulting in <0.001% survivors (Fagundes et al., 2016). Genetically engineered bacteriocins have been proposed to overcome the narrow range of activity of most bacteriocins. Acuña et al. (2012) reported the construction of a chimerical bacteriocin named Ent35-MccV. This hybrid bacteriocin combines in a single molecule the anti-listerial activity of enterocin CRL35 and the anti-E. coli activity of microcin V. This hybrid wide-spectrum bacteriocin was active against pathogenic strains of L. monocytogenes and E. coli O157:H7 and was effective in controlling the growth of both pathogens in skim milk (Acuña et al., 2015). Go to: Application of Bacteriocin-Producing Bacteria to Dairy Products Despite the recent advances in bacteriocin research for food applications, the use of purified bacteriocins in the dairy industry remains limited. Frequently, the application of a bacteriocin alone does not provide sufficient protection against microbial contamination of dairy products. The high cost of bacteriocin isolation and purification also limits the commercial exploration of new bacteriocins. In addition, the restrictive food legislation of the health regulatory authorities (FDA and EFSA) limits the approval of new bacteriocins as food preservatives and, as a consequence, only two bacteriocins (nisin and pediocin) are currently commercially available. The use of bacteriocin-producing bacteria to control contamination microorganisms is an alternative for the use of purified bacteriocins as food additives. Many LAB genera and species have a long history of apparent safe use and they have been granted the GRAS and QPS status. In this regard, incorporation of such bacteria into foods offers a viable solution for controlling contamination microorganisms (Table ​Table22). In addition, LAB are commonly used as starter cultures in food fermentations. Thus, researchers have explored the in situ production of bacteriocins by adding protective cultures that may grow and produce bacteriocins during the manufacture and storage of dairy foods. Many studies have also focused on the selection and development of bacteriocinogenic cultures as cell lysis-inducing agents to improve cheese maturation and flavor (Beshkova and Frengova, 2012). In addition, the use of bacteriocin-producing LAB has been proposed to prevent late blowing occurring in cheeses. Late blowing defect is a major cause of spoilage in ripened cheeses, resulting in the appearance of texture and flavor defects, due to the ubiquitous presence of Clostridium spores (Gómez-Torres et al., 2015). The most common strategies to reduce Clostridium spores are often not sufficient to prevent late blowing in cheeses (Garde et al., 2011) and the use of bacteriocinogenic LAB emerges as an alternative strategy (Table ​Table33). Table 3 Applications of bacteriocin-producing LAB to prevent gas blowing in cheese caused by Clostridium spp. (2000–present). Bacteriocin-producing strain Application technique Product Features Reference Lactobacillus plantarum TF711 Adjunct to commercial starter culture Cow milk cheese Reduction of 2⋅2 log units of Clostridium spores. González and Zárate, 2015 Lactobacillus reuteri INIA P572 Adjunct to commercial starter culture together with glycerol (required for reuterin production). Cow milk cheese Controlled the growth of Clostridium and prevented the development of late blowing. Gómez-Torres et al., 2014 Lactobacillus gasseri K7 Adjunct to commercial starter culture Semi-hard cheese Failed to reduced Clostridium spores, but delayed blowing of cheeses. Matijasic et al., 2007 Lactococcus lactis IFPL 3593 Adjunct to commercial starter culture Semi-hard cheeses 5 log g-1 reduction in the numbers of spores. Martínez-Cuesta et al., 2010 Lactococcus lactis ssp. lactis IPLA 729 Adjunct to starter culture Vidiago cheese Reduced C. tyrobutyricum by 3 log during ripening Rilla et al., 2003 Lactococcus lactis subsp. lactis INIA 415 Starter culture Ovine milk cheese Outgrowth inhibition of Clostridium spores without altering sensory characteristics. Garde et al., 2011 Streptococcus macedonicus ACA-DC 198 Adjunct to starter culture Kasseri cheese Reduced C. tyrobutyricum by 1.4 log during ripening Anastasiou et al., 2009 Streptococcus thermophilus 580 Starter culture Milk curd No gas production for up to 20 days. Mathot et al., 2003 Open in a separate window Among the various LAB species/strains producing bacteriocins, Lactococcus sp. has gained a particular interest in the biopreservation of dairy foods. Benkerroum et al. (2002) tested the effect of in situ bacteriocin production of L. lactis ssp. lactis against L. monocytogenes, in a traditional fermented milk (lben). They found that L. monocytogenes decreased to below the detectable level within 24 h of storage at 7°C in lben fermented with the bacteriogenic starter culture. Moreover, the pathogen was efficiently inactivated from contaminated samples despite the high level of contamination (107 cfu ml-1) for up to 6 days of storage at 7°C (Benkerroum et al., 2002). The application of nisin-producing Lactococcus sp. in dairy foods which requires LAB starters presents a problem, because the wide spectrum of inhibition associated with nisin includes LAB themselves (Abee et al., 1995). Still, Yamauchi et al. (1996) produced a yogurt by incorporating a nisin-producing strain, L. lactis subsp. lactis, in raw milk. The bacteriocin-producing LAB were killed before the addition of the traditional yogurt cultures, and this resulted in increased storage life of the yogurts by preventing the growth of spoilage bacteria (Yamauchi et al., 1996). Recently, Kondrotiene et al. (2018) reported a reduction in L. monocytogenes when three nisin A producing Lc. lactis strains were added to fresh cheese. However, the reduction on Listeria contamination was limited to 2 log units within 7 days of cheese storage. Likewise, the use of a nisin A-producing Lactococcus diacetylactis in a mixed starter culture was not effective in inhibiting L. innocua growth in Cheddar cheese (Benech et al., 2002). Maisnier-Patin et al. (1992) examined the inhibitory effect of another nisin-producing Lc. lactis on L. monocytogenes in Camembert cheese. In the presence of nisin-producing starter, the numbers of the pathogen decreased until the end of the second week, leading to a reduction of 3 log cfu g-1, but a regrowth of Listeria was observed in cheeses throughout ripening (6 weeks). A reduction of L. monocytogenes counts was observed in raw milk cheese by the use of two nisin-producing Lactobacillus lactis subsp. lactis as starter cultures (Rodrígez et al., 2001). In contrast, the use of nisin-producing strains to control E. coli and L. monocytogenes in Feta and Camembert cheeses has shown limited results (Ramsaran et al., 1998). E. coli O157:H7 survived the manufacturing process of both cheeses and was present in cheese made with nisin-producing strains at the end of 75 days of storage in greater numbers than the initial inoculum. The Feta cheese that contained nisin was the only cheese in which L. monocytogenes remained at the level of the initial inoculum after 75 days of storage. The use of bacteriocinogenic pediococci in dairy products also exhibited limited results due to their inability to ferment lactose (Renye et al., 2011). Mills et al. (2011) tested a plantaricin-producing Lactobacillus plantarum strain as an anti-listerial adjunct in the presence and absence of nisin-producing starters for the manufacture of cheeses. The combination of Lb. plantarum strain (at 108 cfu ml-1) with a nisin producer reduced Listeria to undetectable levels by day 28. Moreover, they found that Lb. plantarum was much more effective at inhibiting Listeria than the nisin producer alone. Carnio et al. (2000) studied the anti-listerial potential of a food-grade strain Staphylococcus equorum, producing a bacteriocin named as micrococcin P1, in soft cheese. A remarkable reduction of L. monocytogenes growth was achieved, but this effect was dependent on the contamination level. In addition, a regrowth of the viable Listeria could be observed after 10–16 days of maturation. Likewise, Dal Bello et al. (2012) detected a reduction of L. monocytogenes by 3 log units in cottage cheese, after incubation with a lacticin-481-producing Lc. lactis strain. However, after an initial period, L. monocytogenes counts increased to values comparable to the control. In addition to natural bacteriocinogenic strains, the heterologous production of bacteriocin by genetically engineered LAB was tested in dairy foods (Leroy and De Vuyst, 2004). A lacticin 3147 transconjugant generated via conjugation of bacteriocin-encoding plasmids was used successfully against L. monocytogenes in cottage cheese (McAuliffe et al., 1999). Reductions of 99.9% were seen in cottage cheese held at 4°C after 5 days. In another study, a lacticin 3147 transconjugant presented also a protective effect when applied to the cheese surface, reducing L. monocytogenes by 3 log units (Ross et al., 2000). In contrast, this protective effect was not evident when a nisin-producing culture was used, possibly due to pH instability (Ross et al., 1999). The use of Enterococcus spp. in foods may represent a risk for consumers and requires a safety assessment by the European food authorities (EFSA, 2007). However, enterococci are found naturally in some dairy foods, as they are used as starter cultures and are often part of the microbiota of artisanal cheeses (Domingos-Lopes et al., 2017). In addition, some strains of bacteriocinogenic enterococci were show to lack many of the virulence determinants (Jaouani et al., 2015). De Vuyst et al. (2003) suggested that Enterococcus species could be safely used in food if virulence genes were absent. Consequently, several studies have also employed enterocin-producing enterococcus in food systems. The inhibitory effect of enterocin-producing enterococci against L. monocytogenes and S. aureus in dairy foods such as milk and cheeses was demonstrated by several authors (Giraffa, 1995; Giraffa and Carminati, 1997; Nunez et al., 1997; Lauková et al., 1999a,b: Lauková et al., 2001). Many enterococcal strains producing enterocins were isolated and found to be effective in controlling contamination in cheeses, without compromising the acid-producing activity of the starter and the organoleptic characteristics of the final product (Khan et al., 2010). Bacteriocinogenic E. faecalis strain was show to reduce L. monocytogenes counts in Manchego cheese by 1 and 2 log units after 7 and 60 days, respectively (Nunez et al., 1997). Although inoculation of milk with E. faecalis strain reduced the rate of acid production in the curd, the flavor and bitterness of the final product were not influenced. Other bacteriocinogenic enterococci have also been reported to reduce L. monocytogenes in dairy foods. Pingitore et al. (2012) investigated two bacteriocinogenic enterococci strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch) isolated from cheeses. Growth of L. monocytogenes was inhibited in Minas cheeses containing E. mundtii up to 12 days of storage at 8°C. This bacterium displayed a bacteriostatic effect since Listeria counts remained similar to the initial inoculum. E. faecium strain was found to be less effective, as the bacteriostatic affect occurred only after 6 days at 8°C. Further studies also demonstrated the inhibitory effect of enterocin-producing enterococci strains against L. monocytogenes in raw milk. Vandera et al. (2017) investigated the use of a multiple enterocin-producing bacterial strains possessing the structural entA, entB, and entP enterocin genes. Some strains exhibited a bacteriostatic effect on L. monocytogenes in raw milk incubated at 37°C for 6 h. When raw milk cultures were further incubated at 18°C, viable populations of the pathogen were reduced slightly (by 0.2–0.4 log cfu ml-1) after 24 h and up to 72 h. Achemchem et al. (2006) studied the effectiveness of an E. faecium strain in controlling L. monocytogenes in goat’s milk and goat’s traditional cheese (jben). Coculture experiments of E. faecium and L. monocytogenes in milk demonstrated that the pathogen was not eliminated, but when the bacteriocinogenic strain was previously inoculated in whole milk and left to grow for 12 h before contamination, Listeria was undetectable after 130 h of coculture. Moreover, addition of the bacteriocinogenic strain to jben cheese contaminated with L. monocytogenes prior to packaging, reduced the number of viable Listeria to undetectable levels, after 1 week of storage at 22°C. Coelho et al. (2014) examined the inhibitory effect of enterocin-producing E. faecalis strains against L. monocytogenes in fresh cheese. Inoculation of milk with bacteriocinogenic E. faecalis strains was shown to reduce L. monocytogenes counts by 3–4 log units in fresh cheese compared to the control. The combination of two enterocin producers optimized the reduction of Listeria counts in fresh cheese, decreasing this pathogen by 4 log cfu g-1 in the first 3 days of storage and by 5 log cfu g-1 on day 7. Bacteriocin-producing LAB strains have also been assessed to improve cheese maturation and flavor. These LAB cultures may induce controlled lysis of starter and/or non-starter LAB (NSLAB) and subsequent intracellular release of proteinases and peptidases, resulting in rapid onset of proteolysis and cheese ripening. Lc. lactis producing lacticin 3147 was shown to accelerate cheese ripening and also prevent late blowing in cheese by the inhibition of clostridia growth (Martínez-Cuesta et al., 2010). Various bacteriocin-producing strains were shown to possess a lytic effect on starter cultures. The use of bacteriocin-producing Lc. lactis ssp. cremoris as a starter adjunct in Cheddar cheese manufacture was show to increase the rate of starter lysis. Cheese manufactured with the bacteriocinogenic adjunct exhibited increased cell lysis and higher concentrations of free amino acids, with associated higher sensory evaluation scores (Morgan et al., 1997). Another bacteriocin-producing starter Lc. lactis (a lacticin 3147 producer) was tested for controlling the proliferation of undesirable microorganisms during cheese manufacture. Cheeses made with lacticin 3147-producing starters exhibited significantly lower levels of NSLAB that remained constant over 6 months of ripening (Ryan et al., 1996). Some strains of lacticin 481-producing Lc. lactis were also tested in cheese production and showed to cause partial lysis of starter lactococcal cultures, which continue to grow at a slower rate (O’Sullivan et al., 2003). As a direct result of starter lysis with concomitant enzyme release in the cheese matrix, these strains may be employed to increase cheese ripening. Additionally, a three-strain starter system was tested on cheese by Morgan et al. (2002). This system comprised a bacteriocin producer which causes the lysis of a second strain (sensitive to bacteriocin) and a third strain resistant to bacteriocin activity, for acid production during cheese manufacture. The experimental cheese made with this three-strain starter system showed an increase in lysis and decrease in bitterness compared to cheeses manufactured without the bacteriocin-producing adjunct (Morgan et al., 2002). Go to: Combining Bacteriocins With Other Hurdles One of the approaches to improve the protective action of bacteriocins is the combination with other hurdles such as chemical additives (such as EDTA, sodium lactate, potassium diacetate, and others), heating, and high-pressure treatments (Egan et al., 2016). Narayanan and Ramana (2013) observed that the use of pediocin in combination with eugenol incorporated into polyhydroxybutyrate films worked in synergized form and provided an effective hurdle preventing food contamination. Other researchers used successfully the mixture of bacteriocins and EDTA in the sensitization of Gram-negative bacteria (Prudêncio et al., 2015). Gram-negative bacteria become sensitive to bacteriocins if the permeability of their outer membrane is compromised with chelating agents, such as EDTA (Chen and Hoover, 2003). Zapico et al. (1998) demonstrated a synergistic effect of the combined use of nisin and the lactoperoxidase system (LPS) to control L. monocytogenes in skim milk. A listericidal effect (5.6 log units lower than the control milk) was observed in treatments containing nisin (10 or 100 IU ml-1) with LPS, after 24 h at 30°C. Moreover, when the two preservatives were added in two steps (LPS was added after 3 h and nisin after 5 h of growth), the difference in L. monocytogenes counts increased by 7.4 log units. Several authors also observed the synergistic effect of bacteriocins after temperature treatments (Prudêncio et al., 2015). Kalchayanand et al. (1992) exposed bacteriocin-resistant bacteria to sub-lethal stresses, either at low or high temperatures, and treated with nisin and pediocin. They found that both bacteriocins were effective in reducing the cell viability. This synergistic effect was also observed in Gram-negative bacteria, which are normally insensitive to these bacteriocins (Boziaris et al., 1998). High pressure processing is a common technique for inactivating microorganisms at room temperature, but this treatment does not ensure the complete inactivation of microorganisms (Prudêncio et al., 2015). Several studies have demonstrated the synergistic effect of bacteriocins such as nisin with high pressure processing on the inactivation of food microorganisms (Garriga et al., 2002; Zhao et al., 2013). It is well documented that the use of bacteriocins in combination with these processing techniques enhances bacterial inactivation (Chen and Hoover, 2003). As an example, Rodriguez et al. (2005) demonstrated the efficacy of the application of reduced pressures combined with bacteriocin-producing LAB to improve cheese safety. Go to: Incorporation of Bacteriocins in Antimicrobial Films and Coatings A common strategy for preservation of foods that are eaten raw or without further cooking is the application of edible films or coatings containing antimicrobial substances. The incorporation of antimicrobial compounds such as bacteriocins in edible coatings and films presents as an interesting alternative for ensuring the control of pathogenic microorganisms in food products (Valdés et al., 2017). Edible coatings and films are composed of thin layers of biopolymers that modify the surrounding atmosphere of foods, forming a barrier between the food and the environment, improve the safety, quality, and functionality of food products without changing organoleptic and nutritional properties (Han, 2003; Valdés et al., 2017). The use of purified bacteriocins or bacteriocin-producing bacteria in the packaging system may be more effective in the inhibition of the growth of pathogenic and/or deterioration microorganisms throughout the extent of the latency phase (Balciunas et al., 2013). Hydrocolloids (proteins and polysaccharides) are the most extensively investigated biopolymers in edible coatings and films applied to cheese. They facilitate the incorporation of functional compounds such as bacteriocins and bacteriocin-producing bacteria and allow an increase in the stability, safety, and shelf-life of dairy foods (Scannell et al., 2000a). To date, few studies have investigated the effectiveness of incorporating bacteriocins and/or bacteriocin-producing LAB in coatings and films applied to dairy products. Some authors observed an inhibition of the growth of pathogenic microorganisms in foods packed with coatings and films containing antimicrobial metabolites synthesized by LAB (Cao-Hoang et al., 2010; da Silva Malheiros et al., 2010; Ercolini et al., 2010; Aguayo et al., 2016; Malheiros et al., 2016) or containing viable LAB in the film/coating matrix (Concha-Meyer et al., 2011; Barbosa et al., 2015). Studies of the effectiveness of incorporating purified bacteriocins in edible coatings show a limited reduction of pathogens such as L. monocytogenes. Cheeses, particularly fresh cheeses, are highly perishable due to their high content in caseins, lipids, and water. The complexity of cheese composition and its manufacture support the development of pathogenic and deteriorating microorganisms that increase the risk of foodborne illness and reduce cheese quality and acceptability (Ramos et al., 2012). By acting as additional hurdle, the application of edible coatings and films with incorporation of bacteriocins may overcome problems associated with post-process contamination, therefore enhancing the safety and extending the shelf-life of the cheese. Cao-Hoang et al. (2010) incorporated nisin in films of sodium caseinate applied in semi-soft cheese and observed a small reduction in L. innocua counts (1.1 log cfu g-1) after a week of storage at 4°C. In another study, the incorporation of nisin and lacticin in cellulose coatings applied to Cheddar cheese reduced levels of L. innocua by 2 log cycles, and S. aureus by 1.5 log cycles (Scannell et al., 2000b). However, other studies have shown an effective reduction on pathogen growth. In Ricotta cheese coated with galactomannan and nisin, the growth of L. monocytogenes was prevented for 7 days at 4°C (Martins et al., 2010). The application of a coating in Port Salut cheese, consisting of tapioca starch combined with nisin and natamycin, reduced L. innocua counts above 10 cfu ml-1 during storage, acting as a barrier to post-process contamination (Resa et al., 2014). Recently, Marques et al. (2017) used a biodegradable film incorporated with cell-free supernatant (CFS) containing bacteriocin-like substances of Lactobacillus curvatus P99, to control the growth of L. monocytogenes in sliced “Prato” cheese. These films containing the bactericidal concentration of CFS were able to control L. monocytogenes for 10 days of storage at 4°C. Go to: Conclusion This review highlights the most recent trends in the use of bacteriocins and bacteriocin-producer bacteria in dairy foods. Bacteriocins, either per se or produced by live bacteria, can be successfully incorporated into dairy products to assure safety, extending shelf-life and preserve quality. The use of purified and concentrated bacteriocins as food additives has been the preferred method due to more efficacy, compared to direct application of bacteriocinogenic cultures. However, the effectiveness of bacteriocins in food systems is often low due to several factors such as adsorption to food components, enzymatic degradation, poor solubility, or uneven distribution in the food matrix. On the other hand, the application of live bacteriocin-producing bacteria into dairy foods may overcome the limitations on the use of purified bacteriocins. The bacteriocinogenic LAB added to dairy products such as yogurts and cheeses will ensure continuous production of bacteriocins throughout maturation and storage, and may be included as starter/adjunct cultures in fermentation. The main difficulty associated with this application is the lack of compatibility between the bacteriocin-producing strain and other cultures required in the fermentation of dairy foods. Studies on application of bacteriocins and/or bacteriocin producers have been focused mainly on cheeses because most discovered LAB bacteriocins were effective against Listeria monocytogenes. This foodborne pathogen is often a great concern in traditional cheeses made from raw milk and as a post-processing contaminant in cheeses made from pasteurized milk. Cheeses are particularly vulnerable to the contamination of L. monocytogenes because they provide the appropriate growth conditions for this pathogen. Listeriosis out-breaks linked to the consumption of contaminated cheeses have been reported worldwide. Therefore, the application of bacteriocins as natural preservatives for improving cheese safety has attracted significant research interest in recent years. In contrast, very limited research has been focused on the application of bacteriocins to the preservation of milk, cream, yogurts, and other dairy products. Although most efforts have been devoted to discover novel bacteriocins with unique properties, more studies are necessary for effective application of bacteriocins in dairy foods in order to understand bacteriocin performance in the complex environment of food matrices. In addition, bacteriocins may be combined with other protection tools as part of hurdle technologies to ensure bio-preservation and shelf-life extension of dairy foods. Their use may lie in the combination with other preservation techniques, or in the incorporation in biofilms and active packaging. In the latter case, further studies are necessary to ensure the adaptation of edible coatings and films to bacteriocin activity and efficacy in dairy products. Go to: Author Contributions All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Go to: Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Go to: Footnotes Funding. This work was supported by Instituto de Investigação e Tecnologias Agrárias e do Ambiente (IITAA). The authors are grateful to Fundação para a Ciência e Tecnologia (FCT) UID/CVT/00153/2013. SR was grateful to “Fundo Regional para a Ciência e Tecnologia” (FRCT) Project M3.1.2/F/011/2011. Go to: References Abee T., Krockel L., Hill C. (1995). Bacteriocins: modes of action and potentials in food preservation and control of food poisoning. Int. J. 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