GENERAL AND FOOD MICROBIOLOGY PART II - L. V. Krasnikova - 2016

TOPIC 10. MICROBIOLOGICAL CONTROL OF BAKERY PRODUCTION

10.1. Microbiological control of grain

Grain microorganisms are represented by various species of Bacteria, Yeasts, and Molds. They include saprophytic, phytopathogenic, and human pathogenic microorganisms. Microorganisms that multiply On the surface of plants without harming them are called epiphytic.

The epiphytic microflora of freshly harvested grain is dominated by bacteria of the species Ervinia herbicola (up to 70–95% of the total bacterial count). Bacteria of the species Pseudomonas fluorescens are present in smaller amounts. When storing heavily dusty grain or grain harvested during a hot, dry period, the proportion of spore-forming bacteria such as Bacillus subtilis, B. mycoides, B. licheniformis, B. megatherium, and others increases significantly. Bacteria of the genera Micrococcus, Sarcina, Lactobacillus, and Proteus may be present on damaged grain.

Yeasts found on the grain surface belong to the genera Saccharomyces, Candida, and Torulopsis and do not significantly affect cereal quality. However, under high humidity conditions, they contribute to grain self-heating and The Development of a so-called "bin" odor.

Molds are a constant accompanying microflora of grain. Their growth leads to a decrease in the dry matter mass of the grain and a substantial decline in its quality. Mold Fungi are divided into field fungi and storage fungi.

The first group includes fungi of the genera Alternaria, Fusarium, and Helminthosporium. The mycelium of these fungi penetrates the grain embryo during the milky-ripe stage and subsequently the endosperm. Their development results in shriveled, spotted grains with a black embryo. The second group of fungi is found on stored grain and does not occur on freshly harvested grain. It includes fungi of the genera Aspergillus, Penicillium, Mucor, Rhizopus, Cladosporium, Trichoderma, Trichothecium, Thamnidium, and others.

Phytopathogenic microorganisms parasitize plants, causing various diseases. Molds cause such grain diseases as ergot, smut, and fusariosis. (A detailed description of these pathogens is provided in the textbook, Part I).

Determination of the total microbial count in grain

Sampling. Spot samples of grain are collected using special devices—grain triers. A spot sample is a grain sample taken from a lot in a single operation from one Location. Spot samples are inspected for uniformity. If they are sufficiently uniform, they are mixed to form a composite sample. Next, an average sample is isolated from the composite sample using a divider or manually.

Performing the analysis. A 10 g analytical sample is weighed out from the average grain sample into sterile Petri dishes or weighing bottles and quickly closed. The sample is transferred into a flask containing 90 cm³ of sterile Water. The flask is thoroughly shaken for 5 min either manually or using a special laboratory shaker. Subsequent dilutions are prepared from the first dilution obtained in this manner. The 10^-2 dilution is used to determine mold fungi, while the 10^-3 and 10^-4 dilutions are used for bacteria. Dilutions are plated in duplicate into two sterile Petri dishes using either the pour plate method (1 cm³ added to the bottom of an empty sterile dish, which is then poured over with cooled nutrient Agar) or the surface spread plate method (0.2–0.5 cm³ spread over The surface of solidified agar in a Petri dish). Meat-peptone agar is used for bacteria, whereas wort agar or Sabouraud medium is used for yeasts. Inoculated dishes are incubated at 25–30 °C for bacteria and at 22–25 °C for yeasts and molds. After 48 h, the number of grown colonies is counted, and microscopic preparations are made from them to identify the dominant microflora of the studied grain.

10.2. Microbiological control of flour

The microorganism content in flour typically depends on their initial count in the grain, grain cleaning Methods, flour grade, and storage conditions. In dry flour, microorganisms are in a state of anabiosis because their growth requires free water. During prolonged storage of flour under good conditions, the total microbial count gradually decreases due to the death of non-spore-forming bacterial forms. When flour moisture exceeds the permissible norm by 1–2%, microbial activity resumes, they begin to multiply, and the flour undergoes microbiological spoilage, manifesting as souring, rancidity, and moldiness.

Determination of the total microbial count in flour

Each batch of flour arriving at the enterprise is first subjected to organoleptic evaluation. If an extraneous moldy odor, sour, or rancid taste is detected, a flour sample is prepared, and the total microbial count is determined using a method similar to grain analysis.

Determination of the spore-forming bacteria count in flour

Spore-forming bacteria of the species Bacillus subtilis (hay bacillus), Bacillus licheniformis, Bacillus mycoides, and certain others cause spoilage in wheat flour bakery products known as ropiness (potato disease). During bread baking, the vegetative Cells of these bacteria are destroyed, but their spores retain viability at a crumb Temperature of 95–97 °C. To determine bacterial spores in flour, either the microbiological method or the trial baking method is used.

Microbiological method. A 10 g sample is weighed from the average flour sample, mixed, and then transferred into a flask with 90 cm³ of sterile water, thus obtaining a 10^-1 dilution. The prepared sample is thoroughly mixed and heated in a water bath at 90–95 °C for 10 min. Then the flask contents are cooled, and a 10^-2 dilution is prepared. Using a pipette, 1 cm³ is taken from the 10^-1 and 10^-2 dilutions and inoculated by the pour plate method into Petri dishes, using meat-peptone agar or Yeast agar with 2% sucrose (medium pH 7.0–7.2) as the nutrient medium. The dishes are incubated at 25–30 °C for 2–3 days, after which the colonies grown on them are counted. If 1 g of flour contains fewer than 200 spore-forming bacteria, it is considered normal; from 200 to 1,000, doubtful; and over 1,000, heavily contaminated with these microorganisms.

Trial baking method. Wheat flour is tested for the presence of spore-forming bacteria from May to October. Dough is kneaded from the tested flour using yeast, and three loaf-shaped breads of equal mass are molded from it. After baking, the bread is cooled, the test samples are wrapped in moistened dense paper, placed in a desiccator, and incubated at 37 °C. After 24 h, the first sample is removed from the incubator, sliced, and organoleptically evaluated for signs of ropiness (specific odor, sticky crumb, appearance of silvery threads when pulled apart). Every subsequent 24 h, the second and third loaves are sliced. If signs of spoilage appear after 72 h, the flour is considered sound. In case of test bread spoilage within 24 h, the flour is heavily contaminated with spore-forming bacteria and is permitted for production with certain restrictions.

10.3. Microbiological control of compressed yeast

Cell/15.html">Microscopy. The quality of compressed yeast is evaluated based on Cell size and uniformity, as well as the presence of extraneous Microorganisms in the microscopic preparation. A loopful of yeast is placed into a test tube with a small amount of sterile water and mixed. A drop of the resulting suspension is applied to a Microscope slide, mixed with methylene blue, covered with a coverslip, and examined using an oil immersion objective. The morphological state of the yeast cells, the percentage of dead cells, and the presence of non-saccharomyces yeasts and bacteria are determined. In good-quality yeast, The amount of extraneous microorganisms should not exceed 10–15%.

Determination of the percentage of Saccharomyces yeasts and extraneous microorganisms

In the event of a decrease in the leavening power or stability of compressed yeast, its microbiological composition and the degree of contamination with extraneous microorganisms are determined. For this purpose, either a simplified or a sophisticated method (the LO VNIIKHP method) is used.

Simplified method. Tenfold dilutions of a 1 g compressed yeast sample (10^-5–10^-7) are prepared and plated at 1 cm³ per dish, using wort agar with chalk and nystatin as the nutrient medium to detect acid-forming bacteria. After

After incubation at 30 °C for 48–72 hours, the Petri dishes are examined, and the number of colonies showing clear zones against a white Background of the medium is counted. The colony count is then multiplied by the corresponding dilution factor.

The advanced method involves inoculating yeast onto several selective media to detect various groups of harmful microorganisms. The inoculation scheme According to the LO VNIIRHP method is presented in Table 10.1.

Class="center">Table 10.1. Inoculation scheme for prepared dilutions of compressed yeast onto selective media (0.1 cm³ per plate)

According to this method, a 1 g sample is taken from the middle of a block of compressed yeast and transferred into a flask containing 100 cm³ of sterile water. After thorough mixing, a series of subsequent dilutions (10^-3-10^-8) is prepared from the initial 10^-2 dilution.

The corresponding dilutions are plated onto the nutrient media specified in Table 10.1. After incubating the inoculated plates at the optimum temperature for each group of microorganisms, the number of grown colonies is counted. The total number of Saccharomyces yeast colonies is taken as 100%, and the percentage of extraneous yeasts, mold fungi, putrefactive, lactic acid, and spore-forming bacteria, as well as coliforms, is determined.

The presence of *Proteus vulgaris* bacteria in yeast is determined using Shukevich's method—by inoculating a drop of the 1st and 2nd yeast dilutions into the Condensation water of sloped meat-peptone agar in test tubes. The cultures are placed in an incubator at 37 °C for 24–48 hours. *Proteus* bacilli are detected by a thin, translucent film covering the entire surface of the agar and climbing up the wall of the test tube.

In high-quality compressed yeast, the presence of extraneous yeast is permitted up to a maximum of 10%, and lactic acid bacteria within 15–35%; putrefactive bacteria, *Proteus*, and coliform bacteria must be entirely absent.

10.4. Microbiological Control of Dough

In addition to Saccharomyces yeast and lactic acid bacteria introduced via compressed liquid yeast and starters, extraneous microorganisms may also be present in dough. They are conventionally divided into three groups:

1. Saprophytic microorganisms that do not affect the dough Fermentation process. These include micrococci and sarcinae originating from the flour.

2. Microorganisms that disrupt the normal fermentation process of dough and impair the quality of the finished bread. These are mainly imperfect yeasts of the genera *Candida* (*Candida utilis*, *C. mycoderma*, *C. guilliermondi*, *C. crusei*), *Torulopsis*, as well as certain bacterial species—*Leuconostoc mesenteroides* and *Bacillus coagulans*. Sources of imperfect yeasts include flour, compressed yeast, and whey. Imperfect fungi reduce the maltase activity of compressed yeast, impair its leavening power, and compete for nutrients with the fermentative microflora. Rye starters contaminated with *Candida* fungi develop an off-odor and bitter aftertaste. The growth of *B. coagulans* can lead to acid accumulation and a cheesy odor. *Leuconostoc mesenteroides* accumulates Polysaccharides, resulting in slime formation in liquid scalded starters.

3. Microorganisms causing microbiological spoilage of finished products.

The qualitative COMPOSITION OF THE dough microflora is determined by plating sample dilutions onto selective media, similarly to the LO VNIIRHP method (see Table 10.1). During dough maturation, monitoring is carried out on the gas-producing capacity of the yeast, The rate of its proliferation, and The activity of lactic acid bacteria.

The gas-producing capacity of yeast in dough, along with its maltase activity and osmotolerance, is determined using the I. K. Yeletsky microgasometric device.

The degree of yeast proliferation is determined using a Goryayev counting chamber: the test material is pre-treated using Gutorov's method with an alkali solution followed by methylene blue staining. The number of yeast cells increases as the dough matures. For instance, upon dough mixing, the yeast cell count is about 70 million per 1 g of dough, reaching 112–117 million by the end of the maturation process; when using liquid yeast, the yeast cell count in the dough is much lower, at approximately 20–25 million per 1 g.

Determination of Lactic Acid Bacteria Activity

The activity of lactic acid bacteria is determined according to the method of G. M. Smirnova and M. P. Yurgenson by observing the reduction rate of the blue-green dye Janus Green B or methylene blue. For this purpose, 20 g of dough is mixed with 40 cm³ of water heated to 40 °C. Two 10 ml samples are taken from the mixture. To one test tube (the test sample), 1 cm³ of a 0.05% aqueous solution of Janus Green B (or methylene blue) is added. The second test tube serves as the control. The test tubes are placed in an incubator at 40 °C. The activity of lactic acid bacteria is determined by the time required for the dye to decolorize: low—90–100 min, high—35–50 min, very high—7–25 min.

10.5. Control of Starters Used in Baking

In the baking industry, starters based on pure cultures of lactic acid bacteria or their combinations are used in The production of wheat and rye bread to improve baking properties and enhance product quality.

Starter microorganisms must meet the following requirements:

✵ they must propagate well in flour media;

✵ they must possess a specific level of enzymatic activity;

✵ they must remain stable during continuous cultivation;

✵ they must exhibit antagonistic activity against technologically harmful microflora;

✵ to synthesize certain Vitamins.

The microbiological composition of sourdoughs for the production of wheat and rye flour products varies; as a rule, they include various strains of yeasts, lactic acid bacteria, and propionic acid bacteria.

Sourdoughs for Wheat Dough

The Use of sourdoughs in wheat bread technology helps prevent ropy bread spoilage, increases the nutritional and Biological value of the finished products, and improves their organoleptic characteristics.

Table 10.2 shows the microbiological composition of sourdoughs used for wheat bakery products.

Table 10.2. Microbiological composition of sourdoughs for wheat dough

Wheat mesophilic sourdough is used to intensify gas production in the dough, shorten fermentation time, and improve the quality parameters of finished products made from wheat flour. This sourdough is prepared by co-cultivating yeasts in a saccharified scalded flour mixture (brew) with mesophilic lactic acid bacteria at a temperature of 28-32 °C.

Complex sourdough is recommended for use in the following cases: to improve the quality of products made from flour with weak gluten, to ensure the microbiological purity of products with added wheat bran, and in accelerated dough preparation methods. The sourdough is prepared in a water-flour medium at a water-to-flour ratio of 3:1 and a cultivation temperature ranging from 30 to 32 °C.

Acidophilic sourdough is used to improve the quality of products made from strong and crumbly gluten flour, in accelerated dough preparation technologies, and in the production of rich bakery products with high sugar and fat content.

Propionic acid sourdough is used to prevent ropy bread spoilage and mold growth. The propionic acid bacteria included in the sourdough are capable of synthesizing vitamin B₁₂, which is involved in hematopoiesis.

Vitamin sourdough is recommended for improving the quality of products made from flour with weak gluten, as well as for increasing the biological value of bakery products by enriching the product with β-carotene, which is synthesized by the yeast Burella armenioka Cб-206. The sourdough is prepared on a saccharified flour brew with a moisture content of 82–85% at 22–25 °C for 5–6 hours.

Ergosterol sourdough enhances the biological value of finished products by increasing their ergosterol content (up to 0.2–0.3 mg/100 g), synthesized by the hybrid yeast strain Saccharomyces cerevisiae 576. The sourdough is prepared by co-cultivating yeast and lactic acid bacteria at 30–32 °C with cyclic extraction and renewal every 4 hours. The use of ergosterol sourdoughs is recommended in ecologically unfavorable regions.

Sourdoughs for Rye Dough

When preparing rye dough, it is necessary to introduce specific acid-producing bacteria and yeasts in the required proportion at the very beginning of the process. The required ratio of yeasts and acid-producing bacteria is achieved by preparing rye dough using various sourdoughs.

Rye bread dough can be prepared using:

✵ thick sourdough;

✵ liquid sourdough without scalded flour;

✵ liquid sourdough with scalded flour;

✵ concentrated yeast-free lactic acid sourdough (LCS).

Thick sourdough is used for preparing dough from whole-grain rye flour and dark rye (obdirnaya) flour, as well as from mixtures of various grades of rye and wheat flour. Thick sourdough should have a moisture content of 48–50%, a leavening capacity of up to 25 min, and an acidity of 13–14 degrees if prepared from whole-grain rye flour, or 11–14 degrees if prepared from dark rye flour. The sourdough is prepared using pure cultures of lactic acid bacteria: Lactobacillus plantarum 63, L. brevis 5, L. brevis 78 combined with the yeast strain Candida milleri "Chernorechensky", grown on malt wort. Lactobacterin is also used to develop thick sourdough; it represents a freeze-dried (lyophilized) biomass of a mixture of the aforementioned lactic acid bacteria strains and a pure culture of the yeast Candida milleri "Chernorechensky" in the form of a washout from the surface of an agar slant. When developing a small mass of thick sourdough (up to 5 kg), three doses of dry lactobacterin are dissolved in 30 cm³ of sterile water with preliminary activation for 3–4 hours at 33–35 °C in a nutrient mixture of 0.45 kg of flour and 0.5 dm³ of water. The activated lactobacterin is thoroughly mixed with a yeast suspension (10 cm³) washed from a test tube with a wort-agar slant, or with a suspension of compressed yeast (1 g in 10 cm³ of water). The microorganisms are introduced into the nutrient mixture of 1.8 kg of flour and 2.19 dm³ of water to obtain 5 kg of phase I sourdough, followed by phases II and III of the propagation cycle while observing technological parameters. The fermented phase III sourdough is transferred to a mixing trough (dezhda) and accumulated to the required amount through refreshments.

Liquid sourdough without scalded flour is used to prepare dough from rye flour and mixtures of various grades of rye and wheat flour. This sourdough should have a moisture content of 70–75%, an acidity of 9–13 degrees, and a leavening capacity of up to 35 minutes. The sourdough technology is divided into two cycles: multi-stage propagation and production. In the propagation cycle, liquid sourdough is developed using a mixture of pure yeast cultures Saccharomyces cerevisiae L-1 and Candida milleri "Chernorechensky" combined with a mixture of liquid cultures of Lactobacillus plantarum 30, L. rhamnosus 26, L. brevis 1, L. fermentum 34, or dry lactobacterin for liquid bread sourdoughs made from a mixture of these same strains. The development of liquid sourdough can begin with a large mass directly in the workshop or with a small mass (5 kg in phase I) in the laboratory.

In the propagation cycle, the sourdough is prepared in a water-flour medium at a rye flour-to-water ratio of 35:60. The fermentation temperature is maintained within 28–30 °C; the duration of the process is 2.5–3 hours, the final acidity of the sourdough is 8.0–8.4 degrees, and the leavening capacity is 30–35 minutes. In this cycle, dry lactobacterin for liquid sourdoughs can be used instead of liquid cultures of lactic acid bacteria. In the production cycle, part of the sourdough is used to prepare rye dough, while the remaining part is directed to produce a new batch of sourdough. Next, a semi-continuous method of cultivating sourdough microorganisms is used in the production cycle, in which a specific volume of sourdough with an acidity of 9–13 degrees is removed every 3–4 hours and an equal volume of fresh nutrient medium is added.

Liquid sourdough with scalded flour. The technology of this sourdough differs from that of liquid sourdough without scalded flour in that a nutrient mixture of flour and scalded flour (brew) is prepared for its propagation, after which the aforementioned microorganism cultures are introduced into it and, after mixing, left to ferment for 7–9 hours until an acidity of 7–9 degrees is reached. The propagation cycle consists of three phases, followed by the production cycle of sourdough preparation. The finished sourdough with scalded flour should have a moisture content of 80–85%, an acidity of 9–12 degrees, and a leavening capacity of up to 30 minutes.

Concentrated lactic acid sourdough (CLAS) is prepared in a propagation cycle using a mixture of lactic acid bacteria strains: L. plantarum 30, L. rhamnosus 26, L. brevis 1, L. fermentum 34, or dry lactobacterin for liquid rye sourdoughs. The sourdough should have a moisture content of 60–70%, a temperature of 37–41 °C, and an acidity of 18–24 degrees. The dough is prepared in two stages (CLAS —» dough) or three stages (CLAS —» sponge —» dough). When kneading dough using CLAS, compressed or liquid yeast is used as biological leavening agents. The preparation of dough using concentrated yeast-free lactic acid sourdough is recommended for enterprises with a two-shift operation mode or for small bakeries producing bread from rye flour or a mixture of various grades of rye and wheat flour for only a few hours a day.

10.6. Microbiological Control of Bakery Products

Detection of surface contamination of bread by coliform bacteria (coliforms). A sterile template measuring (10 x 10 cm) is applied to the surface of the bread, and the inner surface of the square is swabbed with a cotton swab moistened in sterile water. The swab is placed in a test tube with sterile water, from which plating is performed onto the surface of Endo agar in a Petri dish. The presence of coliforms on the surface of the bread is not permitted.

Determination of spore-forming bacteria in bread. A 10-20 g piece is excised from the crumb of wheat bread using a sterile lancet, placed into a sterile flask, filled with 100 см³ of sterile water, and shaken vigorously for 5 min. Tenfold dilutions (10^-1-10^-3) are prepared from the resulting suspension, after

which 1 см³ of each dilution is inoculated into test tubes containing meat-peptone broth. The cultures are incubated in an incubator at a temperature of 37±1 °С for 48 h.

Wheat dough products are sliced into 2-3 cm thick pieces, placed in Petri dishes, and autoclaved at a pressure of 0,15 МПа for 20 min. After cooling, 10 см³ of the microorganism culture grown in meat-peptone broth is applied to the surface of the slices. The dishes containing the slices are placed into desiccators with water at the bottom and kept in an incubator at 37±1 °С for two days. If aerobic spore-forming bacteria are present in the test bread, sliming and darkening of the slices are observed, along with a specific odor. Bread evaluation is performed as follows: bread with a titer of 10^-1 is slightly contaminated with spore-forming bacteria; with a titer of 10^-2 - moderately; and with a titer of 10^-3 - heavily.

Topic assignment:

1. Determine the total microbial count in grain and flour.

2. Determine spore-forming bacteria in flour using a microbiological method.

3. Perform microbiological control of compressed yeast.

4. Count the number of dead and budding cells in compressed yeast using a Goryaev chamber.

5. Determine the rate of yeast proliferation in dough by counting their cells in a Goryaev chamber.

6. Determine the activity of lactic acid bacteria in dough using the Smirnova and Yurgenson method.

7. Familiarize with the starters used in baking and examine their microscopic preparations.

Review Questions

1. List the species of bacteria representing the epiphytic microflora of grain.

2. Which mold fungi are classified as field fungi of grain, and which as storage fungi?

3. What methods are used to determine bacterial spores in flour?

4. What extraneous microorganisms are detected in compressed yeast?

5. What indicators are used for microbiological control of dough?

6. What requirements apply to bread starters?

7. Name the starters for wheat dough and their species composition.

8. Name the starters for rye dough and their species composition.

9. How is microbiological control of finished bakery products carried out?