Microbial community analysis of food-spoilage bacteria in commercial custard creams using culture-dependent and independent methods

Custard cream is made from highly nutritive raw materials such as milk and sugar and is easily spoiled by the multiplication of specific microbial contaminants or residents. However, this spoilage microbial community has not been studied. We determined the spoilage microbiota in commercial custard creams using culture-dependent and independent methods. Using the culture-dependent analysis with various agar media, 185 bacterial colonies and 43 eukaryal colonies were isolated from 7 commercial custard cream products. All bacterial isolates were morphologically, physiologically, and genetically identified as bacilli, staphylococci, lactic acid bacteria, and psychrotrophic gram-negative rods. Using culture-independent molecular analysis, the PCR-denaturing gradient gel electrophoresis technique, spoilage of the commercial custard creams was found to be caused by bacilli, staphylococci, lactic acid bacteria, psychrotrophic gram-negative rods, Anoxybacillus sp., Caurobacter sp., and Streptococcus sp. bacteria. The detected spoilage bacteria were the same species as previously detected in spoiled milk products and shown in other reports, suggesting that spoilage bacteria in a raw material easily grow in processed foods made from milk. We determined the spoilage microbial communities in commercial custard creams, and these are the first data concerning spoilage microbiota in nonfermented processed foods using a culture-independent analysis. Our study will be useful for the manufacture and safe preservation of dairy products because the first step toward safe food preservation by food manufacturers is to understand the spoilage microbiota in a target food to select optimal preservatives and to reduce the use of food additives.     

A health-sanitary evaluation of lacteal desserts for consumption in Santa Cruz de Tenerife

The consumption of lacteal desserts in the province of Santa Cruz de Tenerife is notably high. However, there are no legal standards in Spain regarding microbiological quality. For this reason, we have decided that it would be of interest to carry out a health-sanitary study of these products, with the aim of discovering their microbial content. 330 samples of lacteal desserts on sale in the province of Santa Cruz de Tenerife have undergone analysis. They have been divided into three groups: cream caramel (egg and vanilla) (80), mousse (60) and the third group, known as “other desserts”, which includes custard and the rest of lacteal desserts not included in the previous groupings (190). Neither E. coli, Salmonella spp., Shigella spp., nor Staphylococcus aureus have been detected in any of the samples analysed. In spite of the fact that the results obtained do not reflect high microbiological contamination, we consider it necessary to lay down legal standards, with reference values, for these lacteal products, which will guarantee good microbiological quality.     

SUBJECT: BITTY CREAM AND RELATED PROBLEMS:PROBLEMS ASSOCIATED WITH BACTERIAL SPORES IN HEAT-TREATED MILK AND DAIRY PRODUCTS

Spore spoilage in pasteurized milk and UHT and sterilized milk is reviewed with particular reference to cream. The problem of the incidence of bitty cream and the effectiveness of refrigeration in checking this is discussed. Results of studies on the bacterial quality of bulk raw milk and tanker reload milk using the thermoduric count are given in detail. Current methods for pasteurizing cream are described and studies on the effect of storage temperature on the shelf life of pasteurized cream are referred to in detail with particular reference to the lag in bacterial multiplication and the use of the Methylene Blue Test.     

Characterization of Cucumber Fermentation Spoilage Bacteria by Enrichment Culture and 16S rDNA Cloning

Commercial cucumber fermentations are typically carried out in 40000 L fermentation tanks. A secondary fermentation can occur after sugars are consumed that results in the formation of acetic, propionic, and butyric acids, concomitantly with the loss of lactic acid and an increase in pH. Spoilage fermentations can result in significant economic loss for industrial producers. The microbiota that result in spoilage remain incompletely defined. Previous studies have implicated yeasts, lactic acid bacteria, enterobacteriaceae, and Clostridia as having a role in spoilage fermentations. We report that Propionibacterium and Pectinatus isolates from cucumber fermentation spoilage converted lactic acid to propionic acid, increasing pH. The analysis of 16S rDNA cloning libraries confirmed and expanded the knowledge gained from previous studies using classical microbiological methods. Our data show that Gram‐negative anaerobic bacteria supersede Gram‐positive Fermincutes species after the pH rises from around 3.2 to pH 5, and propionic and butyric acids are produced. Characterization of the spoilage microbiota is an important first step in efforts to prevent cucumber fermentation spoilage. Practical Application An understanding of the microorganisms that cause commercial cucumber fermentation spoilage may aid in developing methods to prevent the spoilage from occurring.     

Effects of gassericins A and T, bacteriocins produced by Lactobacillus gasseri, with glycine on custard cream preservation

Lactobacillus gasseri LA39 and LA158 isolated from human-infant feces produce bacteriocins named gassericins A and T, respectively. Both gassericins have high heat stability (121°C, 10 min), good pH tolerance (pH 2-11), and strong bactericidality against many gram-positive bacteria, especially lactic acid bacteria, and thus are expected to be effective food preservatives. A microwell plate assay against 12 strains of custard cream spoilage bacteria showed that the gassericins had broader antibacterial spectra than nisin A. Although the gassericins allowed gram-negative isolates to grow, they successfully inhibited the growth of all tested bacterial strains in microwells with the addition of glycine. Glycine was bacteriostatic against many strains except lactic acid bacteria. For practical use, gassericin A was efficiently produced by cultivation in a food-grade medium improved using cheese whey, nourishing proteose peptone, and surfactant yolk lecithin. The practical preservative effect of gassericin A and glycine was verified from the viability of 4 isolated strains, Bacillus cereus, Lactococcus lactis ssp. lactis, Achromobacter denitrificans, and Pseudomonas fluorescens, in custard creams. Custard cream containing 123 arbitrary units of gassericin A per milliliter entirely growth-inhibited the 2 gram-positive strains. In custard cream containing an insufficient amount of gassericin A (49 arbitrary units/mL), the gram-positive strains gradually grew but were completely inhibited by the addition of 0.5% (wt/wt) glycine. The 2 gram-negative strains did not multiply even in the additive-free custard cream, probably because of the unsuitable growth environment. This is the first report showing the combined effect of bacteriocin and glycine and their application for food preservation, which may be helpful for future use in the food industry.     

The shelf-life of dairy products: 2. Raw milk and fresh products

In the second of this series of articles, the specific factors controlling the shelf-life of raw milk, pasteurized milk and cream, cottage cheese and yogurt are considered. The shelf-life of raw milk depends on the quality of the milk ex-farm and on subsequent transport and storage conditions. Shelf-life may be enhanced by deep cooling or thermization. Post heat treatment contamination by Gram negative psychrotrophic bacteria is the major determinant of shelf-life of pasteurized products. In cultured dairy products, yeast and mould contamination is the most usual cause of spoilage.     

冷鲜牛肉贮藏中菌群结构及优势菌致腐性的分析

为探究牛肉在0?℃冷藏下微生物菌群变化和优势腐败菌,采用培养依赖的16S rRNA结合高通量测序技术分析牛肉样品的微生物多样性变化。通过定期测定接种牛肉汁的生长、感官、蛋白酶活性、pH值和挥发性盐基氮（total volatile basic nitrogen,TVB-N）值,分析分离株的致腐特征。结果表明,牛肉冷藏中感官品质保持良好,15?d出现异味,18?d有腐臭味。而样品中菌落总数第3天快速上升,15?d菌落总数为8.73（lg（CFU/g））,之后呈现稳定,假单胞菌（Pseudomonas spp.）、热死环丝菌（Brochothrix thermosphacta）、肠杆菌（Enterobacter）和乳酸菌4 种分离培养基中细菌与菌落总数增长趋势相似,其中假单胞菌增长最快,肠杆菌数和乳酸菌数生长最慢。两种菌群鉴定结果显示,冷鲜牛肉初始微生物构成复杂,包括热死环丝菌、不动杆菌属（Acinetobacter spp.）、气单胞菌属（Aeromonas spp.）和假单胞菌等多种菌属构成,而腐败末期菌群构成趋于单一,假单胞菌和热死环丝菌为优势腐败菌,特别是莓实假单胞菌（P. fragi）。将腐败分离株10?株假单胞菌、4?株热死环丝菌和1?株蜂房哈夫尼菌（Hafnia alve）接种于冷藏的牛肉汁,发现假单胞菌和蜂房哈夫尼菌的接种组感官评分、pH值和TVB-N值高于热死环丝菌,且假单胞菌有较强的蛋白酶活性。研究表明,结合感官和微生物评价冷鲜牛肉贮藏期为15?d,且菌群多样性下降,假单胞菌和热死环丝菌是优势腐败菌,其中假单胞菌致腐性较强。     

MICROBIOLOGICAL QUALITY OF SELECTED STREET FOODS IN PORT HARCOURT, NIGERIA

The microbiological quality of selected ready‐to‐eat foods sold on the streets of Port Harcourt, Nigeria, was evaluated. Twenty to twenty‐eight samples each of 10 different types of foods were analyzed. About 50% of the foods were deep fat fried, 20% were baked, 10% each were either boiled, pasteurized and frozen, or fermented and chilled products. Methods of food preparation and handling practices influenced the shelf lives of the foods, and their implications on microbiological quality and safety are discussed. The heterotrophic counts were highest for yogurt and ice cream and lowest for chin‐chin and buns. Bacterial counts for pancake, moi moi, cake, meat pie and egg roll on McConkey agar gave comparable results with counts obtained on Deoxycholate agar. Eleven bacteria genera were isolated. Bacillus spp. and Klebsiella spp. occurred in most of the foods, followed by Staphylococcus spp. and Micrococcus spp., while other species exhibited a random distribution. Generally, the foods were microbiologically safe despite defective handling practices.     

Development of Molecular Typing Methods for Bacillus spp. and Paenibacillus spp. Isolated from Fluid Milk Products

Bacillus spp. and related sporeformers are important food spoilage organisms. While use of molecular subtyping methods has provided important information on the ecology and transmission of foodborne pathogens, the lack of rapid, reliable, and affordable subtyping methods for Bacillus spp. has limited our ability to understand and control their transmission throughout the food chain. We used a previously described collection of Bacillus spp. and Paenibacillus spp. isolated from dairy products to develop a DNA sequencing‐based subtyping approach for these spoilage microorganisms. After optimization of polymerase chain reaction (PCR) parameters, primers targeting the rpoB housekeeping gene allowed for successful amplification in all isolates. rpoB sequencing allowed differentiation of 29 subtypes (that is, sequence types) among the 57 isolates characterized. Phylogenetic analyses of rpoB sequences revealed distinct monophyletic lineages that correlated with bacterial genera (Bacillus and Paenibacillus) as well as with species or species‐like assemblages within each genus. rpoB sequencing provided improved subtype discrimination over 16S rDNA sequencing; therefore, rpoB sequencing allows for both sensitive subtype discrimination as well as for species and genus identification. Analysis of subtypes isolated over time in dairy products revealed the presence of both persistent and transient bacterial subtypes, indicating that application of these methods can improve our understanding of the ecology of these spoilage organisms and can help in identification of bacterial niches that may contribute to the persistence of these spoilage organisms in food systems.     

THE DEVELOPMENT OF TAINTS IN PASTEURIZED CREAM

A survey of 231 samples from 11 brands of cream showed that the keeping quality of pasteurized cream depends on the development of both bacteriological and non-bacteriological taints. The water agar test is suggested as a simple test that is able to predict bacterial spoilage and to identify correctly 95 per cent of those creams with a keeping quality at 5^oC of not less than 14 days. Spoilage that developed within three days was due to bacterial action in only 57 per cent of the samples.     

Development of a Monte Carlo simulation model to predict pasteurized fluid milk spoilage due to post-pasteurization contamination with gram-negative bacteria

Psychrotolerant gram-negative bacteria introduced as post-pasteurization contamination (PPC) are a major cause of spoilage and reduced shelf life of high-temperature, short-time pasteurized fluid milk. To provide improved tools to (1) predict pasteurized fluid milk shelf life as influenced by PPC and (2) assess the effectiveness of different potential interventions that could reduce spoilage due to PPC, we developed a Monte Carlo simulation model that predicts fluid milk spoilage due to psychrotolerant gram-negative bacteria introduced as PPC. As a first step, 17 gram-negative bacterial isolates frequently associated with fluid milk spoilage were selected and used to generate growth data in skim milk broth at 6°C. The resulting growth parameters, frequency of isolation for the 17 different isolates, and initial concentration of bacteria in milk with PPC, were used to develop a Monte Carlo model to predict bacterial number at different days of shelf life based on storage temperature of milk. This model was then validated with data from d 7 and 10 of shelf life, collected from commercial operations. The validated model predicted that the parameters (1) maximum growth rate and (2) storage temperature had the greatest influence on the percentage of containers exceeding 20,000 cfu/mL standard plate count on d 7 and 10 (i.e., spoiling due to PPC), which indicates that accurate data on maximum growth rate and storage temperature are important for accurate predictions. In addition to allowing for prediction of fluid milk shelf life, the model allows for simulation of “what-if” scenarios, which allowed us to predict the effectiveness of different interventions to reduce overall fluid milk spoilage due to PPC through a set of proof-of-concept scenario (e.g., frequency of PPC in containers reduced from 100% to 10%; limiting distribution temperature to a maximum of 6°C). Combined with other models, such as previous models on fluid milk spoilage due to psychrotolerant spore-forming bacteria, the data and tools developed here will allow for rational, digitally enabled, fluid milk shelf life prediction and quality enhancement.     

Tracking spore-forming bacterial contaminants in fluid milk-processing systems

The presence of psychrotolerant Bacillus species and related spore formers (e.g., Paenibacillus spp.) in milk has emerged as a key biological obstacle in extending the shelf life of high-temperature, short-time pasteurized fluid milk beyond 14 d. A recently developed rpoB DNA sequence-based subtyping method was applied to characterize spoilage bacteria present in raw milk supplies for 2 processing plants, and to assess transmission of these organisms into pasteurized products. Thirty-nine raw milk samples and 11 pasteurized product samples were collected to represent the processing continuum from incoming truck loads of raw milk to packaged products. Milk samples were held at 6°C for up to 16 d and plated for bacterial enumeration at various times throughout storage. Among the 88 bacterial isolates characterized, a total of 31 rpoB allelic types representing Bacillus and Paenibacillus spp. were identified, including 5 allelic types found in both raw milk and finished product samples. The presence of the same bacterial subtypes in raw and commercially pasteurized milk samples suggests that the raw milk supply represents an important source of these spoilage bacteria. Extension of the shelf life of high-temperature, short-time pasteurized fluid milk products will require elimination of these organisms from milk-processing systems.     

Traceback Investigation for Salmonella Contamination at Egg Processing Plants in South Korea: Prevalence,Antibiotic Resistance,and Epidemiological Tracing by Rep‐PCR Fingerprinting

We conducted a survey of Salmonella from 8 egg‐breaking plants and a farm to determine the prevalence and the source of the bacteria. The contents of 2400 shell eggs (20 eggs per pool), 75 pasteurized liquid egg products, and 120 unpasteurized liquid egg products from 8 egg‐breaking plants in South Korea were examined. In liquid egg samples, 4 Salmonella‐positive samples from 120 unpasteurized ones (3.3%) and 5 positive samples from 75 pasteurized ones (6.7%) were identified; no eggs were positive for Salmonella among shell egg samples. To trace the source of Salmonella, we revisited the 2 Salmonella‐positive plants (plants A and C). We investigated the equipment and environments of the plants and a henhouse (farm A) that supplied shell eggs to plant A, and collected additional liquid eggs and shell eggs from plants A and C. All Salmonella isolates from plant A and the associated farm A, except for a single Typhimurium strain from farm A, were serotyped as Bareilly. Three serovars, including one Bareilly, four Tennessee, and one Richmond, were isolated from plant C. Most Salmonella isolates were susceptible to tested antibiotics. To identify differences between isolates, molecular subtyping by using the automated rep‐PCR system was conducted. All Salmonella Bareilly (S. Bareilly) strains from plant A exhibited high similarity, indicating possible contamination by Salmonella strains from the henhouse A. Meanwhile, 2 S. Bareilly strains from plant C, one from liquid egg at the 1st visit and the other from container at the 2nd visit, exhibited identical antibiotic resistance and similar subtyping pattern, but clearly discriminated from the ones of plant A.     

Gas‐producing and spoilage potential of Enterobacteriaceae and lactic acid bacteria isolated from chilled vacuum‐packaged beef

This study aimed to enumerate and identify lactic acid bacteria and Enterobacteriaceae from spoiled and nonspoiled chilled vacuum‐packaged beef and determine their potential to cause blown pack spoilage. These microbial groups were also enumerated in nonspoiled samples and detected in abattoir samples. The potential of isolates to cause ‘blown pack’ spoilage of vacuum‐packaged beef stored at chilled temperature (4 °C) and abuse temperature (15 °C) was investigated. Populations of lactic acid bacteria in exudate of spoiled and nonspoiled samples were not significantly different (P > 0.05), whereas the number of lactic acid bacteria on the surface was significantly higher (P < 0.05) in spoiled samples as compared to nonspoiled samples. The population of Enterobacteriaceae species in exudate and on the surface of samples were significantly higher (P < 0.05) in spoiled packs in comparison with nonspoiled packs. Results of the deterioration potential showed that ‘blown pack’ spoilage was noticeable after 7 days at 15 °C and after 6 weeks at 4 °C for samples inoculated with Hafnia alvei.     

Psychrotrophs in dairy products: Their effects and their control

Health concerns and technological effects of psychrotrophic bacteria in dairy products are reviewed, as well as methods to control their presence and development. The various Gram‐negative and Gram‐positive psychrotrophic species are listed and, with respect to pathogenic psychrotrophs, emphasis is given on Listeria monocytogenes, Yersinia enterocolytica, and Bacillus cereus. The influence of psychrotrophic bacteria on the quality of raw milk, pasteurized and UHT milks, butter, ice cream, cheese, and powders is examined. Public health considerations of Listeria monocytogenes, Yersinia enterocolytica, and Bacillus cereus of these various dairy products are also presented. Methods that can be used to eliminate or control the development of psychrotropic bacteria include low or high temperatures, chemicals, gases, the lactoperoxidase system, lactic acid bacteria, microfiltration, bactofugation, lactoferrin‐related proteins, sanitation, flavors, and naturally occurring spore germinants.     

Instrumental Method for Characterizing Protein Foams

A whipping machine for cream analysis (Cream Tester CTII) was used to foam egg white, milk proteins and soy protein isolate. Foam formation and final rigidity were characterized by the current input to the beating motor. In addition, specific volume, rigidity (compressive strength by Instron) and stability (drainage) of the foams were determined by methods that minimized foam damage during handling. Current input during whipping distinguished fresh vs pasteurized egg white and positively correlated with compressive strength of final egg white foams. Such correlation was not found for milk protein and soy protein isolate foams of lower strength and stability.     

Beer enemy number one: genetic diversity,physiology and biofilm formation of Lactobacillus brevis

Lactobacillus brevis is the most significant beer spoilage bacteria worldwide. It is found as a contaminant at all stages of brewing, including during primary and secondary fermentation, storage, filtration and the packaging process. In production with flash pasteurisation and subsequent hygienic filling, avoiding and tracing secondary contaminations is the key to a microbiologically stable product. However, L. brevis strains vary in their spoilage potential and can grow in many different beer types. This study presents a physiological test scheme for growth potential and biofilm formation in various media. It was determined that a large number of L. brevis strains can form biofilms as a first coloniser. The identification of the species alone is therefore not enough to be sure of the spoilage risk, which shows the need for a more in depth differentiation. DNA fingerprint techniques are crucial to differentiate isolates of this species at strain level. The rep‐PCR fingerprint system (GTG)₅ was used to differentiate a selected collection of 20 isolates, which were characterised in growth and biofilm formation in various media. The data showed a high variation within the selected isolates. As second step, generated fingerprint clusters of L. brevis were traced back to contamination sources in a German brewery, revealing a high number of isolates with potentially varying growth, spoilage and biofilm potential. L. brevis being the demonstrator species, the PCR system used is a powerful and compatible tracing and troubleshooting tool for all kinds of spoilage bacteria in the brewing industry. © 2019 The Institute of Brewing & Distilling     

Bacterial Quorum Sensing and Food Industry

Abstract: Food spoilage and biofilm formation by food‐related bacteria are significant problems in the food industry. Even with the application of modern‐day food preservative techniques, excessive amounts of food are lost due to microbial spoilage. A number of studies have indicated that quorum sensing plays a major role in food spoilage, biofilm formation, and food‐related pathogenesis. Understanding bacterial quorum‐sensing signaling systems can help in controlling the growth of undesirable food‐related bacteria. This review focusses on the various signaling molecules produced by Gram‐negative and Gram‐positive bacteria and the mechanism of their quorum‐sensing systems, types of signaling molecules that have been detected in different food systems using biosensors, the role of signaling molecules in biofilm formation, and significance of biofilms in the food industry. As quorum‐sensing signaling molecules are implicated in food spoilage, based on these molecules potential, quorum‐sensing inhibitors/antagonists can be developed to be used as novel food preservatives for maintaining food integrity and enhancing food safety. Practical Application: Bacteria use signaling molecules for inter‐ and intracellular communication. This phenomenon of bacterial cell‐to‐cell communication is known as quorum sensing. Quorum‐sensing signals are implicated in bacterial pathogenicity and food spoilage. Therefore, blocking the quorum‐sensing signaling molecules in food‐related bacteria may possibly prevent quorum‐sensing‐regulated phenotypes responsible for food spoilage. Quorum‐sensing inhibitors/antagonists could be used as food preservatives to enhance the shelf life and also increase food safety.     

MICROBIOLOGICAL SHELF‐LIFE STUDIES ON COMMERCIALLY MANUFACTURED YEAST

ABSTRACT

During three replicate studies, cream, compressed and dry yeast samples were stored for 21 days at 4, 10, 25 and 37C. At 3‐day intervals, bacteria were quantified using standard plate counting procedures, and 1,044 predominant colonies isolated and characterized. The highest counts of aerobic bacteria and Enterococcus spp., up to 7 and 8 log colony‐forming units (cfu) per mL or g, were found when cream and compressed yeast samples were exposed to elevated temperatures (25 and 37C), while lower counts (4 and 6 log cfu/mL or g) were obtained from refrigerated samples (4C). At 10C, counts of aerobic bacteria and Enterococcus spp. increased from 4 to 7 log cfu/mL or g, highlighting the importance of temperature control during the storage and distribution of perishable cream and compressed yeast. Vacuum‐packaging of dry yeast reduced the growth of aerobic populations. Throughout storage, Lactobacilli dominated the bacterial populations in both cream and compressed yeast (45 to 78%), while Enterococcaceae predominated in vacuum‐packaged dry yeast (54 to 68%), suggesting they were primarily responsible for the spoilage of commercial yeast products.

PRACTICAL APPLICATIONS

In addition to producing high quality yeast for baking and brewing, it is an important objective for yeast manufacturers to increase product shelf life. Temperatures chosen for this shelf‐life study simulated various elevated temperatures of practical use for yeast processors and downstream consumers. For example, 4C (recommended refrigeration temperature), 10C (potential abuse temperature during transportation), 25C (wine‐making temperature) and 37C (temperatures often encountered in bakeries). Results from our study showed that storage temperature and time considerably influenced bacterial growth patterns associated with commercially manufactured fresh yeast. Bacterial spoilage and sensory deterioration were observed during lengthened storage periods, and especially at higher storage temperatures. Even when stored at 10C, cream and compressed yeast samples became bacteriologically and visually spoiled. It was therefore apparent that any break in the cold chain during processing, storage or distribution could affect the development of spoilage bacteria resulting in shortened product shelf life of commercially manufactured fresh yeast. Conversely, this study found vacuum‐packaged dry yeast to be the most bacteriologically stable manufactured product.     

冷藏海鲈鱼优势腐败菌的筛选和鉴定

分离鉴定4 ℃冷藏条件下海鲈鱼的优势腐败菌,通过选择性培养基筛选获得单一菌株,对各菌株进行致腐能力的测定,确定冷藏海鲈鱼的优势腐败菌。对冷藏海鲈鱼的优势腐败菌进行菌落形态观察及部分生理生化实验、16S rDNA分子鉴定。结果表明,有4 株冷藏海鲈鱼优势腐败菌,其中1 株为草莓假单胞菌（Pseudomonas fragi）,1 株为腐败希瓦氏菌（Shewanella putrefaciens）,其余2 株为假单胞菌（Pseudomonas sp.）。在4 ℃冷藏条件下,草莓假单胞菌的致腐能力最强,其次是腐败希瓦氏菌和假单胞菌。