Use In Biopreservation Research Articles

Use of Live Biopreservatives and Bacteriophages to Enhance the Safety of Meat Products

Critical health considerations for both raw and processed meats include addressing bacterial spoilage and ensuring safety. Nitrites and nitrates are widely used in the meat industry to enhance color and flavor and extend shelf life. However, health concerns linked to their use make reducing nitrites and nitrates in meat production a significant challenge with potential benefits for both the food industry and consumer health. This challenge has been addressed with the use of biopreservatives, i.e., substances extracted from natural sources or produced by fermentation that can enhance food quality and safety. In this article, we assess the use of live biopreservatives (LBs), defined here as microorganisms that produce antimicrobial substances that can be used to preserve and extend the shelf life of food. Moreover, the potential synergistic effects of LBs with bacteriophages and biodegradable food packaging for meat is also explored. This innovative combination offers a comprehensive approach to meat preservation, enhancing both microbial control and sustainability. Overall, the inclusion of LBs extends the shelf life of meat products through bacteriostatic mechanisms, whereas bacteriophages offer direct (lytic) action against pathogens. Enhancing meat preservation and safety with mixed microbe-mediated strategies requires deeper empirical and theoretical insights and further revision of laws and ethical considerations.

Chemical composition of sausages processed with starter cultures Lactobacillus curvatus and Lactococcus lactis subsp. lactis during storage in vacuum packaging

Sausages belong to the category of cooked meat products with a short shelf life, which is associated with their rapid spoilage and unsuitability for consumption. One of the ways to extend the shelf life of sausages is the use of biopreservatives, including cultures of lactic acid bacteria. The aim of the study was to determine the chemical composition and organoleptic characteristics of "Juicy" sausages processed with a starter culture of Lactobacillus curvatus or a mixture of starter cultures Lactobacillus curvatus + Lactococcus lactis subsp. lactis before vacuum packaging and storage in a chilled state. Storage of sausages in the control variant in vacuum packaging in a chilled state for up to 18 days did not affect the moisture, protein, fat, and ash content, but on the 21st day, their spoilage was observed in the form of appearance of cloudy juice, sliminess, sour odor, and delamination of the vacuum packaging. Processing sausages with a starter culture of Lactobacillus curvatus or a mixture of starter cultures Lactobacillus curvatus + Lactococcus lactis subsp. lactis before vacuum packaging did not affect the moisture, protein, fat, and ash content up to 30 days of storage in a chilled state. On the 36th day, spoilage of sausages was observed in both variants of treatment with lactic acid bacteria cultures, characterized by the appearance of cloudy juice, sour odor, and delamination of the vacuum packaging. The pH value of sausages in both variants of treatment with lactic acid bacteria cultures decreased throughout the storage period and reached a level of 6.05 and 5.91 units on the 30th day. Processing sausages with either a single culture or a mixture of lactic acid bacteria cultures extends their shelf life in a chilled state by 12 days. The obtained results may justify the use of microbial biopreservatives for the production of cooked sausage products, which will help reduce losses due to spoilage during storage in vacuum packaging in a chilled state. The use of a mixture of lactic acid bacteria cultures for processing sausages requires further research and may be effective in case of detection of aerobic spoilage microorganisms.

Optimization of Enzyme-Assisted Extraction of Bioactive Compounds from Sea Buckthorn (Hippophae rhamnoides L.) Leaves: Evaluation of Mixed-Culture Fermentation.

Hippophae rhamnoides L. leaves possess a remarkable amount of polyphenols that could serve as a natural remedy in various applications. In comparison, numerous techniques, such as conventional and high-pressure techniques, are available for extracting the bioactive fractions from sea buckthorn leaves (SBL). However, enzyme-assisted extraction (EAE) of SBL has not been comprehensively studied. The aim of this study was to optimize critical EAE parameters of SBL using the cellulolytic enzyme complex, Viscozyme L, to obtain a high-yield extract with a high concentration of bioactive compounds. In order to determine the optimal conditions for EAE, the study employed a central composite design and response surface methodology to analyze the effects of four independent factors (pH, temperature, extraction time, and enzyme concentration) on two different responses. Our findings indicated that under optimal conditions (3:15 h extraction, temperature 45 °C, pH 4.9, and 1% Viscozyme L v/w of leaves DW), EAE yielded 28.90 g/100 g DW of the water-soluble fraction. Furthermore, the EAE-optimized liquid extract was continuously fermented using an ancient fermentation starter, Tibetan kefir grains, which possess lactic acid bacteria (LAB) and have significant potential for use in biopreservation. Interestingly, the results indicated various potential prebiotic characteristics of LAB. Additionally, alterations in the cell wall morphology of the SBL residue after EAE were examined using scanning electron microscopy (SEM). This study significantly optimized EAE parameters for sea buckthorn leaves, providing a promising natural source of bioactive compounds for various applications, such as nutraceuticals, functional foods, and high-value products.

Synergistic Effect of Combination of Various Microbial Hurdles in the Biopreservation of Meat and Meat Products-Systematic Review.

The control of spoilage microorganisms and foodborne pathogens in meat and meat products is a challenge for food producers, which potentially can be overcome through the combined use of biopreservatives, in the form of a mix of various microbial hurdles. The objective of this work is to systematically review the available knowledge to reveal whether various microbial hurdles applied in combination can pose an effective decontamination strategy for meat and meat products. PubMed, Web of Science, and Scopus were utilized to identify and evaluate studies through February 2023. Search results yielded 45 articles that met the inclusion criteria. The most common meat biopreservatives were combinations of various starter cultures (24 studies), and the use of mixtures of non-starter protective cultures (13 studies). In addition, studies evaluating antimicrobial combinations of bacteriocins with other bacteriocins, BLIS (bacteriocin-like inhibitory substance), non-starter protective cultures, reuterin, and S-layer protein were included in the review (7 studies). In one study, a biopreservative mixture comprised antifungal protein PgAFP and protective cultures. The literature search revealed a positive effect, in most of the included studies, of the combination of various bacterial antimicrobials in inhibiting the growth of pathogenic and spoilage bacteria in meat products. The main advantages of the synergistic effect achieved were: (1) the induction of a stronger antimicrobial effect, (2) the extension of the spectrum of antibacterial action, and (3) the prevention of the regrowth of undesirable microorganisms. Although further research is required in this area, the combination of various microbial hurdles can pose a green and valuable biopreservation approach for maintaining the safety and quality of meat products.

Efficacy of Bacteriocin Producing Lactic Acid Bacteria Isolated from Ugba and Okpiye Against some Selected Bacterial Food Spoilage Agents

Numerous food products owe their production and characteristics to the fermentative activities of microorganisms. Bacteriocins are antimicrobial proteins produced by bacteria that kill or inhibit the growth of other related species. Lactic acid producing bacteria were isolated from Ugba and Okpiye using standard methods. The test organisms used were Staphylococcus aureus, Pseudomonas aeruginosa (VRFPA04) and Escherichia coli (ST2747) Fifty (50) samples of Ugba and Okpiye were collected and evaluated for the presence of bacteriocin lactic acid producers using a selective medium; De Mann Rogosa Sharpe (MRS) agar. A total of 288 isolates was isolated from the two traditional food condiments; 148 from ugba and 140 from Okpiye. It was observed that the total lactic acid bacterial count in Ugba ranges from 5.80 x 104 to 1.7 x 107 cfu/ml on MRS medium; while that of Okpiye ranges from 5.0 x 104 to 2.0 x 107 cfu/ml on MRS medium. Lactic acid bacterial count was higher in Ugba than in Okpiyeconsidering the mode of storage in the market. The highest inhibitory activity was demonstrated against E. coli in Ugba sample while the least activity was demonstrated against P. aeuroginosa, also a significant high level of inhibition was shown against S. aureus while P. aeuroginosa and E. coli had little or no inhibitory activities recorded for Okpiye samples. The bacteriocin producers isolated from Ugba and Okpiye (U5, U6, OK1, OK2, OK3 and OK4) were identified as Lactobacillus sp and Lactococcus sp based on their morphological and biochemical characteristics. The antibacterial activity of the bacteriocins produced by the lactic acid bacteria has potential for use in biopreservation of condiments against food spoilage agents.

The Biochemical Alteration of Enzymatically Hydrolysed and Spontaneously Fermented Oat Flour and Its Impact on Pathogenic Bacteria.

Avena sativa (A. sativa) oats have recently made a comeback as suitable alternative raw materials for dairy substitutes due to their functional properties. Amylolytic and cellulolytic enzyme-assisted modifications of oats produce new products that are more appealing to consumers. However, the biochemical and functional alteration of products and extracts requires careful selection of raw materials, enzyme cocktails, and technological aspects. This study compares the biochemical composition of different A. sativa enzyme-assisted water extracts and evaluates their microbial growth using spontaneous fermentation and the antimicrobial properties of the ferment extracts. Fibre content, total phenolic content, and antioxidant activity were evaluated using traditional methodologies. The degradation of A. sativa flour was captured using scanning electron microscopy (SEM); moreover, sugar and oligosaccharide alteration were identified using HPLC and HPLC-SEC after INFOGEST in vitro digestion (IVD). Additionally, taste differentiation was performed using an electronic tongue with principal component analysis. The oat liquid extracts were continuously fermented using two ancient fermentation starters, birch sap and Tibetan kefir grains. Both starters contain lactic acid bacteria (LAB), which has major potential for use in bio-preservation. In fermented extracts, antimicrobial properties against Gram-positive Staphylococcus aureus and group A streptococci as well as Gram-negative opportunistic bacteria such as Escherichia coli and Pseudomonas aeruginosa were also determined. SEM images confirmed the successful incorporation of enzymes into the oat flour. The results indicate that using enzyme-assisted extraction significantly increased TPC and antioxidant activity in both the extract and residues. Additionally, carbohydrates with a molecular mass (MM) of over 70,000 kDa were reduced to 7000 kDa and lower after the incorporation of amylolytic and cellulolytic enzymes. The MM impacted the variation in microbial fermentation, which demonstrated favourable antimicrobial properties. The results demonstrated promising applications for developing functional products and components using bioprocessing as an innovative tool.

Bacteriocin: A new strategic antibiofilm agent in food industries

Contamination of food products with microorganisms from various sources is a serious concern as it not only affects the food quality also leads to spoilage and therefore may cause several illnesses. Chemical preservatives may not be considered as a potential way as it has many disadvantages. The use of bio-preservatives can replace it. Bacteriocins are recently drawing attraction to be used in food products because of several benefits. These are antibiotics that can act against pathogenic microorganisms. It is absolutely safe to use them and can be easily digested in the human body. Bacteriocins are of three types and each one has a specific mechanism of action against biofilms. Biofilms are found in the extracellular matrix of different colonizing species and in the food industry they can be formed easily in different conditions. In specific conditions, bacteriocins cannot function against microorganisms and to enhance their functions hurdle technology is found highly suitable. It has been observed that the use of hurdle technology can promote the activity of bacteriocins to a greater extent. The incorporation of bacteriocins into packaging films can protect the food product from spoilage. There are several ways to insert bacteriocin into packaging material. This method is a great option to enhance the shelf life of food products by reducing the number of spoilage organisms. Further study is required to understand its mechanism of action more vividly which may help to use them as a bio preservative in the food industry in a broader spectrum.

Bacteriocin, Antimicrobial as A New Natural Food Preservative: Its Potential and Challenges

Improving food safety and quality remains a major challenge in worldwide. Modern society tends to choose the foods that are fresh tasting, free of chemical additives, rich in nutrients and has been introduced to minimally processed. Bacteriocins as new natural preservatives could be produced by Gram-positive bacteria and Gram-negative bacteria, even though it is mostly originated from Lactic Acid Bacteria (LAB). Bacteriocin-producing bacteria as well as bacteriocin has long been used in some countries as alternatives to replace chemical additives. The use of these antimicrobial agents has been proven effective in food manufacturing and distribution chain, especially in increasing shelf life of food products. Other potential from the use of bio-preservatives in various types of food is still huge. However, approval of bio-preservatives and their commercially applications is still limited. This study will review the selection criteria for bacteria used, the mechanism of action, its potential application in the food and pharmaceutical industries and its potential as an anti-cancer agent. In addition, this study will explore the main challenges faced including its stability and the possible risk of resistance to microorganisms. This review will also describe a series of processes for identification, characterization, and approval of the use of bacteriocins before they are commercially marketed. In addition, the safety issue of using bacteriocin-producing bacteria, especially as probiotics, also needs to be considered as a fundamental for quality control and the improvement of drug and food safety.

Application of bacterial cellulose film as a biodegradable and antimicrobial packaging material

Nowadays, conventional petroleum-based plastic had become the third-largest solid waste volume in municipal solid waste. Although recycling plastic was aimed to minimize its quantity, the recycling process still releases toxic materials into the environment. Thus, the best solution is to use biodegradable plastic. Additionally, due to the health concern of consumers, especially over the safety of food, the packaging industry had shown greater interest in the use of bio-preservatives for antimicrobial (AM) packaging. This study aimed to develop a new biodegradable and AM packaging material from bacterial cellulose (BC) film. BC is a natural biopolymer synthesized by bacteria. Here, BC was produced through a static fermentation of Acetobacter xylinum 0416 in the Hestrin-Schramm medium, while the AM packaging was developed using a soaking method. The BC film produced was incorporated with lauric acid (LA) at different concentration (2, 4 and 6% w/v) and different soaking time (4, 14 and 24 hr). The characteristics and functionalities of the incorporated BC-LA film were analyzed for the AM activity and biodegradation test. The results depicted that incorporation of BC film with LA provides a good inhibition effect towards the growth of Bacillus subtilis, while the effect on the growth of Escherichia coli was absent. Additionally, the pure BC film also goes through more than 50% of degradation in 3 days and 100% of degradation starting from day 7 in the soil. Further investigation had determined that the responsible degrading microbes were Bacillus sp. and Rhizopus sp. As overall, this new packaging material was successfully developed and showed wide potential applications to be used in food, pharmaceutical and medical field.

Microbial Spoilage and Preservation of Wine: Using Weapons from Nature's Own Arsenal -A Review

The winemaking process includes multiple stages at which microbial spoilage can occur, altering the quality and hygienic status of the wine and rendering it unacceptable. The major spoilage organisms include species and strains of the yeast genera Brettanomyces, Candida, Hanseniaspora, Pichia, Zygosaccharomyces etc., the lactic acid bacterial genera Lactobacillus, Leuconostoc, Pediococcus, etc. and the acetic acid bacterial genera Acetobacter and Gluconobacter. The faults caused include bitterness and off.flavours (mousiness, ester taint, phenolic, vinegary, buttery, geranium tone), and cosmetic problems such as turbidity, viscosity, sediment and film formation. These spoilage organisms can also affect the wholesomeness of wine by producing biogenic amines and precursors of ethyl carbamate. The judicious use of chemical preservatives such as sulphur dioxide (S02) during the winemaking process decreases the risk of microbial spoilage, but strains vary considerably in their S02 sensitivity. There is, moreover, mounting consumer bias against chemical preservatives, and this review focuses on the possible use of biopreservatives in complying with the consumers' demand for clean and green products.

Diversity of Streptococcus thermophilus in bacteriocin production; inhibitory spectrum and occurrence of thermophilin genes

The bacteriocin-producing Streptococcus thermophilus strains that can dominate in natural dairy ecosystems, may also enhance safety in products obtained from natural cultures. In this study, we sought to identify bacteriocin production and bacteriocin genes in 75 strains of dairy and plant origin. The strains were tested for antimicrobial activity against pathogens or pathogen models, spoiling bacteria, and lactic acid bacteria associated with dairy products. All strains moderately inhibited Staphylococcus aureus P310, none inhibited Listeria innocua LMG 11387T or Clostridium tyrobutyricum LMG 1285T. In addition, 14 were active against one or more indicators in addition to S. aureus P310. Inhibition of other starter bacteria was more common than the inhibition of unwanted microorganisms. The involvement of a proteinaceous compound was ascertained in all cases. Results suggested that the selection of bacteriocinogenic S. thermophilus strains for use in biopreservation must take into account the effects exerted on other lactic acid bacteria.PCR detection of thermophilin genes proved unreliable in predicting antimicrobial activity. For S. thermophilus PRI36 and PRI45, with relevant inhibitory features, the identity of the bacteriocin genes present in the thermophilin 9 cluster was defined, thus revealing novel variants for this genome region.

Isolation of bacteriocin - producing lactic acid bacteria from 'Ugba' and 'Okpiye', two locally fermented nigerian food condiments

In this work, 100 samples each of 'ugba' and 'okpiye' were evaluated for the presence of bacteriocin producing lactic acid bacteria. Thirty strains showing antibacterial activity against at least one of the indicator organisms were selected from a total of 752 colonies isolated from the condiments. Out of the 30, only five strains retained activity after the pH of the broth supernatant was adjusted to 6.5. When evaluated by the agar-well diffusion assay, the spectra of inhibitory activity showed that Staphylococcus aureus was the most sensitive indicator organism tested, while Listeria monocytogenes was the most resistant. One strain (UG 2) was active against Escherichia coli. The assays using the cell-free supernatant of the cultures showed that the bacteriocins were completely inactivated by the proteolyses as well as by the chloroform treatment. In ethanol, the activity of the compounds was only partially modified. When incubated in a water bath at 80°C for 30 min, no significant activity loss was recorded. The antimicrobial activity of the bacteriocins produced by the lactic acid bacteria has potential for use in biopreservation of condiments against the spoilage and food - borne pathogens.

Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.

Selection and evaluation of seafood-borne psychrotrophic lactic acid bacteria as inhibitors of pathogenic and spoilage bacteria

In this study, inhibitory psychrotrophic lactic acid bacteria were isolated and investigated for future use in biopreservation of seafood products. Screening of 5575 colonies isolated from various seafood products resulted in the selection of 132 colonies presenting inhibitory properties. Among them, 52 isolates had characteristics of LAB and showed growth at 15 °C but not at 30 °C. The inhibition spectrum of these 52 isolates against 14 target strains (Gram-positive and -negative) showed inhibition of typical seafood spoiling and pathogenic bacteria and enabled the formation of seven interesting clusters. Sequencing of the 16S rRNA gene of a representative isolate from each cluster identified three Leuconostoc gelidum, two Lactococcus piscium, one Lactobacillus fuchuensis and one Carnobacterium alterfunditum. Theses strains did not produce histamine nor tyramine, and showed no particular antibiotic resistance profile. Growth rate as a function of temperature was tested for one L. piscium and one L. gelidum isolate and confirmed their psychrotrophic behavior. One out of seven isolates showed bacteriocin-like activity. The inhibition mechanisms of the other isolates are still unknown but may be due to competition for substrate. Absence of a bacteriocin-like component could be a positive point to gain rapid authorization for food application in France. This collection of LAB is now ready for testing on products.

The Use of Biopreservatives in the Control of Bacterial Contaminants of Sugarcane Alcohol Fermentation

ABSTRACT: Efficacy and safety of biopreservatives were studied for microbiological control of alcohol fermentation. Minimum inhibitory concentrations of nisin and lysozyme, and combinations with EDTA and Tween 20, were determined for Lactobacillus plantarum, L. fermentum, and L. confusus. The highest MIC value for nisin was 9.0 ppm for L. fermentum. Lysozyme was efficient against L. plantarum (700 ppm). A 24 factorial design was used to study the effect of biopreservatives and potentiators on the growth kinetics of Lactobacillus fermentum. The combination of 8.6 ppm of nisin plus 0.1% of Tween 20 delayed the lag phase by 12 h. The biopreservatives did not affect the fermentation kinetics of Saccharomyces cerevisiae.

Biopreservation by lactic acid bacteria.

Biopreservation refers to extended storage life and enhanced safety of foods using the natural microflora and (or) their antibacterial products. Lactic acid bacteria have a major potential for use in biopreservation because they are safe to consume and during storage they naturally dominate the microflora of many foods. In milk, brined vegetables, many cereal products and meats with added carbohydrate, the growth of lactic acid bacteria produces a new food product. In raw meats and fish that are chill stored under vacuum or in an environment with elevated carbon dioxide concentration, the lactic acid bacteria become the dominant population and preserve the meat with a "hidden' fermentation. The same applies to processed meats provided that the lactic acid bacteria survive the heat treatment or they are inoculated onto the product after heat treatment. This paper reviews the current status and potential for controlled biopreservation of foods.